Method of treating or preventing ras-mediated diseases

ABSTRACT

Disclosed are compounds, for example, a compound of formula I, 
     
       
         
         
             
             
         
       
     
     wherein R, R 0 , R 1 -R 8 , n, X, Y, Y′, and E are as described herein, pharmaceutical compositions containing such compounds, and methods of treating or preventing a disease or condition for example, cancer, mediated by the ras gene.

CROSS-REFERENCE TO A RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.14/571,690, filed Dec. 16, 2014, the disclosure of which is incorporatedherein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with partial support under NIH/NCI Grant NumbersCA 155638 and CA 148817. Therefore, the U.S. Government has certainrights in this invention.

BACKGROUND OF THE INVENTION

Cancer is a leading cause of death in the developed world, with over onemillion people diagnosed and more than 500,000 deaths per year in theUnited States alone. Overall it is estimated that at least one in threepeople will develop some form of cancer during their lifetime. There aremore than 200 different histopathological types of cancer, four of which(breast, lung, colorectal, and prostate) account for over half of allnew cases in the U.S. (Jemal et al., Cancer J. Clin., 53, 5-26 (2003)).

Many of these tumors arise from mutations that activate Ras proteins,which control critically important cellular signaling pathways thatregulate growth and other processes associated with tumorigenesis. Thename “Ras” is an abbreviation of “Rat sarcoma” reflecting the way thefirst members of the Ras protein family were discovered. The name “ras”also is used to refer to the family of genes encoding these proteins.

Ras-driven cancers have remained the most intractable diseases to anyavailable treatment. New therapeutic and preventative strategies areurgently needed for such cancers (Stephen et al., Cancer Cell, 25,272-281 (2014)). Drug discovery programs worldwide have soughtRas-selective drugs for many years, but heretofore no avail (Spiegel, etal., Nature Chem. Biol., 10, 613-622 (2014)). New drugs that selectivelytarget abnormal or mutant Ras and/or Ras-mediated pathological processesin patients' tumors will enable highly efficacious treatments of suchpatients while minimizing toxicity to cells and tissues with normal Rasfunctions (Stephen et al., supra; Spiegel et al., supra).

Ras proteins are key regulators of several aspects of normal cell growthand malignant transformation, including cellular proliferation, survivaland invasiveness, tumor angiogenesis and metastasis (Downward, NatureRev. Cancer, 3, 11-22 (2003)). Ras proteins are abnormally active inmost human tumors due to mutations in the ras genes themselves, or inupstream or downstream Ras pathway components, or other alterations inRas signaling. Targeted therapies that inhibit Ras-mediated pathwaystherefore are expected to inhibit the growth, proliferation, survivaland spread of tumor cells having activated or mutant Ras. Some such newexperimental therapeutic agents have shown promising activity inpreclinical studies, albeit with only modest activity in human clinicaltrials.

Genetic mutations in ras genes were first identified in human cancerover 3 decades ago. Such mutations result in the activation of one ormore of three major Ras protein isoforms, including H-Ras, N-Ras, orK-Ras, that turn on signaling pathways leading to uncontrolled cellgrowth and tumor development. Activating ras gene mutations occur denovo in approximately one third of all human cancers and are especiallyprevalent in pancreatic, colorectal, and lung tumors. Ras mutations alsodevelop in tumors that become resistant to chemotherapy and/orradiation, as well as to targeted therapies, such as receptor tyrosinekinase inhibitors (Gysin et al., Genes Cancer, 2, 359-372 (2011)). Whileras mutations are relatively infrequent in other tumor types, forexample, breast cancer, Ras can be pathologically activated by certaingrowth factor receptors that signal through Ras.

Although ras gene mutations have been known for many years, therecurrently are no available cancer therapeutics approved by the U.S. Foodand Drug Administration that are known to selectively suppress thegrowth of tumors driven by activated Ras. In fact, Ras has beendescribed as “undruggable” because of the relative abundance in cellsand high affinity for its substrate, GTP (Takashima and Faller, ExpertOpin. Ther. Targets, 17, 507-531 (2013)).

In addition to its role in cancer, activated Ras is important in avariety of other diseases, collectively referred to as “rasopathies.”One such disease, neurofibromatosis type 1 (NF1), a very prevalentautosomal dominant heritable disease, is caused by a mutation inneurofibromin, a Ras GAP (inactivating protein), which results in Rashyperactivation in the relatively common event of loss of the second NF1allele. Such mutations reportedly affect 1:3000 live births. The mostdire symptoms associated with NF1 include numerous benign tumors(neurofibromas) arising from precursor nerve cells and Schwann cells ofthe peripheral nervous system. These tumors can cause severe problemsdepending on their location within the body, such as hearing or visionloss, as well as disfiguring masses on visible areas. Less common butextremely serious complications may arise when central nervous systemgliomas develop or plexiform neurofibromas become transformed, resultingin the development of metastatic peripheral nerve sheath tumors (Tidymanand Rauen, Curr. Opin. Genet. Dev., 19, 230-236 (2009)). Another raredevelopmental disease which is attributable to hyperactive H-Ras isCostello syndrome. This condition causes a range of developmentalabnormalities as well as predisposing patients to a variety of benignand malignant neoplasms (Tidyman and Rauen, supra).

Several approaches to treat diseases that arise from activating rasmutations have been undertaken. Because full maturation of the Rasprotein requires lipid modification, attempts have been made to targetthis enzymatic process with inhibitors of farnesyl transferase andgeranylgeranyltransferase, but with limited success and significanttoxicity. Targeting of downstream components of Ras signaling withinhibitors of Raf/Mek/Erk kinase components of the cascading pathway hasbeen an extremely active area of pharmaceutical research, but alsofraught with difficulties and paradoxes arising from complex feedbacksystems within the pathways (Takashima and Faller, supra).

Inhibitors targeting components within the PI3K/Akt pathway also havenot been successful as single agents, but presumably might synergizewith Raf/Mek/Erk pathway inhibitors to block Ras-dependent tumor growthand survival. Similarly, several other molecular targets have beenidentified from RNAi screening, which might provide new opportunities toinhibit the growth of Ras-driven tumors; such other potential targetsinclude CDK4, Cyclin D1, Tiam1, Myc, STK33, and TBK, as well as severalgenes involved in mitosis (Takashima and Faller, supra).

The nonsteroidal anti-inflammatory drug, sulindac (FIG. 1) has beenreported to selectively inhibit proliferation of cultured tumor cellshaving ras mutations (Herrmann et al., Oncogene, 17, 1769-1776 (1998)).Extensive chemical modifications of sulindac and the related NSAID,indomethacin, have been aimed at removing cyclooxygenase-inhibitoryactivity, while improving anticancer activity (Gurpinar et al., Mol.Cancer Ther., 12, 663-674 (2013); Romeiro et al., Eur. J. Med. Chem.,44, 1959-1971 (2009); Chennamaneni et al., Eur. J. Med. Chem., 56, 17-29(2012)). An example of a highly potent antiproliferative derivative is ahydroxy-substituted indene derivative of sulindac, OSIP-487703 (FIG. 1),that was reported to arrest colon cancer cells in mitosis by causingmicrotubule depolymerization (Xiao et al., Mol. Cancer Ther., 5, 60-67(2006)). OSIP-487703 also was reported to inhibit the growth and induceapoptosis of human SW480 colon cancer cells. These properties of mitoticarrest and microtubule disruption were shared by several additionalrelated compounds, including a pyridine (CP461) and trimethoxy (CP248)substituted variants (FIG. 1) (Lim et al., Clin. Cancer Res., 9,4972-4982 (2003); Yoon et al., Mol. Cancer Ther., 1, 393-404 (2002)).However, there was no reported association of antitumor properties ofthese compounds (FIG. 1) with Ras function, but rather such propertieswere attributed to direct binding to the microtubule subunit, tubulin,thereby causing mitotic arrest and blocking cell division. Still otherreports describe their ability to induce apoptosis by inhibition of cGMPphosphodiesterase (Thompson et al., Cancer Research, 60, 3338-3342(2000)).

Other investigators reported that sulindac sulfide (FIG. 1) can inhibitRas-induced malignant transformation, possibly by decreasing the effectsof activated Ras on its main effector, the c-Raf-1kinase, due to directbinding to the ras gene product p21 in a non-covalent manner (Herrmannet al., supra). Sulindac sulfide also can inhibit focus formation, amarker of malignant transformation, by rat or mouse fibroblasts byforced Ras expression, but not by other transformation pathways (Gala etal., Cancer Lett., 175, 89-94 (2002); Herrmann et al., supra). Sulindacsulfide was reported also to bind Ras directly and interfere withnucleotide exchange. Several groups additionally reported that sulindacinterferes with Ras binding to the downstream signaling kinase c-Raf,and blocks activation of downstream signaling or transcription (Herrmannet al., supra; Pan et al., Cell Signal., 20, 1134-1141 (2008)).

The aforementioned findings led to efforts to improve the Ras inhibitoryactivity of sulindac sulfide through chemical modifications (Karaguni etal., Bioorg. Med. Chem. Lett., 12, 709-713 (2002)). Several derivativeswere identified that were more potent inhibitors of tumor cellproliferation, and four related compounds (FIG. 2) exhibited selectivitytowards a Ras-transfected MDCK cell line compared to the parental cellline. Three of these compounds also potently disrupted the Ras-Rafinteraction. However, none of the four were more potent toward themutant K-Ras-bearing SW-480 cell line, although they did inhibit Erkphosphorylation and bound weakly to the G-domain of H-Ras (Waldmann etal., Angew. Chem. Int. Ed. Engl., 43, 454-458 (2004)).

In addition to sulindac sulfide, the non-COX inhibitory sulfonemetabolite of sulindac has been reported to have selective effects ontumor cells with mutant Ras. For example, transfection of Caco-2 colontumor cells with the activated K-Ras oncogene caused cells treated witheither sulindac sulfide or sulfone to undergo apoptosis earlier thannon-transfected cells. (Lawson et al., Cancer Epidemiol. BiomarkersPrev., 9, 1155-62 (2000)). Other investigators have reported thatsulindac sulfone can inhibit mammary tumorigenesis in rats and that theeffect was greater on tumors with the mutant H-Ras genotype (Thompson etal., Cancer Research 57, 267-271 (1997)). However, other investigatorsreport that the inhibition of colon tumorigenesis in rats by eithersulindac or sulindac sulfone occurs independently of K-Ras mutations (deJong et al., Amer. J. Physio. Gastro and Liver Phys. 278, 266-272(2000)). Yet other investigators report that the K-Ras oncogeneincreases resistance to sulindac-induced apoptosis in rat enterocytes(Arber et al., Gastroenterology, 113, 1892-1900 (1997)). As such, theinfluence of Ras mutations on the anticancer activity of sulindac andits metabolites is controversial and unresolved, and has not beenexploited to improve anticancer potency or selectivity.

Certain other compounds have been described with selective toxicitytoward cells expressing activated Ras. A high-throughput phenotypicscreen of over 300,000 compounds was conducted within NIH MolecularLibraries Screening Center program to identify compounds which weresynthetically lethal to cells expressing oncogenic H-Ras. A leadcompound, ML210 (FIG. 3), inhibited growth of cells expressing mutantRas with an IC50 of 71 nM, and was 4-fold selective versus cells lackingoncogenic Ras. Though the specific molecular target of ML210 is unknown,the compound was chemically optimized to eliminate reactive groups andimprove pharmacologic properties (ML210, Dec. 12, 2011 update, ProbeReports from NIH Molecular Libraries Program, Bethesda,http://www.ncbi.nlm.nih.gov/books/NBK98919/).

A separate high-throughput screen identified two compounds, RSL3 andRSL5 (FIG. 3) which induce non-apoptotic, Mek-dependent, oxidative celldeath (Yang and Stockwell, Chem. Biol., 15, 234-245 (2008). RSL5, like apreviously identified Ras synthetic lethal compound, erastin (FIG. 3),binds the voltage-dependent anion channel (VDAC) (Dolma et al., CancerCell, 3, 285-296 (2003)). Yet another small-molecule screen identifiedoncrasin, a compound selectively active against K-Ras mutant cell lines(Guo et al., Cancer Res., 68, 7403-7408 (2008)). One analog, NSC-743380(FIG. 3), is highly potent and has shown anti-tumor activity in apreclinical model of K-Ras driven renal cancer (Guo et al., PLoS One, 6,e28487 (2011)). A prodrug approach has recently been described foroncrasin derivatives, to improve stability, pharmacokinetics, and safety(Wu et al., Bioorg. Med. Chem., 22, 5234-5240 (2014)). A syntheticlethal screen using embryonic fibroblasts derived from mice expressingthe oncogenic K-Ras (G12D) identified a compound, lanperisone (FIG. 3),that induced non-apoptotic cell death via a mechanism involvingoxidative stress (Shaw et al., Proc. Natl. Acad. Sci. USA, 108,8773-8778 (2011)). In contrast to the synthetic lethal approach, afragment-based screening approach paired with crystallographic studieshas been used to identify compounds which irreversibly bind to andinhibit K-Ras in lung tumor cells having the relatively rare G12C rasgene mutation (Ostrem et al., Nature, 503, 548-551 (2013)). Whilecompounds of this series potently inhibit Ras through a covalentinteraction, the low frequency of this mutation may limit the utility ofsuch compounds. Finally, a new investigational strategy for targetingoncogenic Ras has been described (Zimmerman et al., J. Med. Chem., 57,5435-5448 (2014)) which involves structure guided design and kineticanalysis of benzimidazole inhibitors targeting the PDEδ prenyl bindingsite.

WO 97/47303 and WO 2014/047592, U.S. Patent Application Publication Nos.2003/0009033 and 2003/0194750, U.S. Pat. Nos. 6,063,818, 6,071,934;5,965,619; 5,401,774; 6,538,029; and 6,121,321, and U.K. Patent No. GB1370028 disclose certain anticancer compounds; however, these documentsdo not disclose that the compounds have Ras-specific activity, nor anybasis for a selective Ras-directed method of use.

The foregoing shows that there exists an unmet need for a method oftreating preventing or preventing Ras-dependent diseases or undesirableconditions.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a method of inhibiting a human ornonhuman mammalian Ras-mediated biological process, which methodcomprising administering in vivo or in vitro a Ras-inhibitory amount ofat least one compound of formula I, a pharmaceutically acceptable saltor prodrug thereof:

wherein:

R and R₀ are independently selected from hydrogen, hydroxyl, alkyl,trifluoromethyl, amino, alkoxy and alkylamino, or R and R₀ together isoxygen or sulfur, or R and R₀ together is a single-bonded or adouble-bonded nitrogen bonded to one or more of hydrogen, hydroxyl,alkyl, and trifluoromethyl; n is 0, 1 or 2;

R₁, R₂, R₃, and R₄ are independently selected from hydrogen, hydroxyl,halogen, alkyl, trifluoromethyl, alkoxy, and alkylmercapto;

R₅, R₆, R₇, and R₈ are independently selected from hydrogen, alkyl,trifluoromethyl and alkoxy; or R₅ and R₆ together form a carbon-carbonbond;

Y is hydrogen, alkyl, or trifluoromethyl, and Y′ is hydrogen, alkyl,trifluoromethyl, amino, alkylamino, or alkoxy, or Y and Y′ together isoxygen or sulfur, or Y and Y′ together is a single-bonded or adouble-bonded nitrogen bonded to one or more of hydrogen, hydroxyl,alkyl, and trifluoromethyl;

X is selected from hydrogen, alkyl, trifluoromethyl, alkoxy,alkylmercapto, and hydroxyl with the proviso that X is not hydroxyl whenY and Y′ together is oxygen in a compound of formula I wherein R₅ and R₆together is a carbon-carbon bond, or X is NR′R″, where R′ is selectedfrom the group consisting of hydrogen, hydroxyl, alkyl, trifluoromethyl,alkoxy, alkenyl, alkynyl, hydroxyalkyl, polyhydroxyalkyl,alkylaminoalkyl, dialkylaminoalkyl, aminoalkyl, aryl, arylalkyl,arylalkenyl, arylcycloalkyl, arylcycloalkenyl, carbocyclyl, andcarbocycloalkyl where the carbocycle of the carbocyclyl and thecarbocycloalkyl is selected from 7-membered carbocyclic rings containingno double bond, or one, two or three double bonds, 6-memberedcarbocyclic rings containing no double bond, or one or two double bonds,5-membered carbocyclic rings containing no double bond, or one or twodouble bonds, 4-membered carbocyclic rings containing no double bond orone double bond and 3-membered carbocyclic rings containing no doublebond, heterocyclyl, and heterocycloalkyl, where the heterocycle of theheterocyclyl and heterocycloalkyl is selected from 7-memberedheterocyclic rings, 6-membered heterocyclic rings, and 5-memberedheterocyclic rings, and the aryl of the aryl, arylalkyl, arylalkylenyl,arylcycloalkyl, or arylcycloalkenyl structure or the carbocyclic orheterocyclic structure may optionally be substituted with one or more ofhalo, alkyl, trifluoromethyl, hydroxyl, alkoxy, amino, alkylamino,dialkylamino, mercapto, alkylmercapto, carboxamido, aldehydo, cyano,oxo, alkylcarbonyloxy, and sulfonamido; and R″ is selected fromhydrogen, alkyl, hydroxyalkyl, aminoalkyl, dialkylaminoalkyl,cyanoalkyl, haloalkyl, alkylcarbonylalkylcarbonyloxy, pyridyl, and COR₁₁wherein R₁₁ is selected from hydrogen, amino, alkyl, trifluoromethyl,alkoxy, alkylmercapto, and aryl; or R′ and R″ together form a 5-, 6- or7-membered, saturated or unsaturated, heterocyclic ring containing atleast one nitrogen, and optionally oxygen and/or sulfur, and theheterocyclic ring may optionally be substituted with one or more ofhalo, alkyl, trifluoromethyl, hydroxyl, alkoxy, amino, alkylamino,dialkylamino, mercapto, alkylmercapto, carboxamido, aldehydo, cyano,oxo, alkylcarbonyloxy, and sulfonamido; and

E is a substituted or unsubstituted, saturated or unsaturated,7-membered, 6-membered, 5-membered, 4-membered or 3-membered carbocyclicor heterocyclic ring;

wherein at least one compound of formula I or a pharmaceuticallyacceptable salt or prodrug thereof is administered alone or incombination with at least one additional therapeutic agent other than acompound or pharmaceutically acceptable salt or prodrug of formula I.

In one aspect, when R and R₀ are hydrogen, n is 1, three of R₁, R₂, R₃,and R₄ are hydrogens and one is halogen, alkyl, or alkoxy or two of R₁,R₂, R₃ and R₄ are hydrogens and two are alkoxy, R₅ and R₆ together forma carbon-carbon bond, R₇ is hydrogen, R₈ is alkyl, Y and Y′ together isoxygen, X is NR′R″ wherein R″ is hydrogen, and R′ is a substituted aryl,then E cannot be a substituted aryl. For example, when R and R₀ arehydrogen, n is 1, three of R₁, R₂, R₃, and R₄ are hydrogens and one ishalogen, alkyl, or alkoxy or two of R₁, R₂, R₃ and R₄ are hydrogens andtwo are alkoxy, R₅ and R₆ together form a carbon-carbon bond, R₇ ishydrogen, R₈ is alkyl, Y and Y′ together is oxygen, X is NR′R″ whereinR″ is hydrogen, R′ is an aryl substituted with any of halo, alkoxy,amino, alkylamino, dialkylamino and sulfonamido, then E cannot be asubstituted aryl wherein two substituents are identically selected fromhydroxyl and alkoxy.

The present invention further provides a method of therapeutically orprophylactically treating a human or nonhuman mammalian patient with adisease or condition treatable by inhibition of one or more Ras-mediatedbiological process, which method comprising administering to a patientin need thereof a therapeutically or prophylactically effective amountof at least one ras-inhibitory compound of formula I, a pharmaceuticallyacceptable salt or prodrug thereof:

wherein:

R and R₀ are independently selected from hydrogen, hydroxyl, alkyl,trifluoromethyl, amino, alkoxy and alkylamino, or R and R₀ together isoxygen or sulfur, or R and R₀ together is a single-bonded or adouble-bonded nitrogen bonded to one or more of hydrogen, hydroxyl,alkyl, and trifluoromethyl; n is 0, 1 or 2;

R₁, R₂, R₃, and R₄ are independently selected from hydrogen, hydroxyl,halogen, alkyl, trifluoromethyl, alkoxy, and alkylmercapto;

R₅, R₆, R₇, and R₈ are independently selected from hydrogen, alkyl,trifluoromethyl and alkoxy; or R₅ and R₆ together form a carbon-carbonbond;

Y is hydrogen, alkyl, or trifluoromethyl, and Y′ is hydrogen, alkyl,trifluoromethyl, amino, alkylamino, or alkoxy, or Y and Y′ together isoxygen or sulfur, or Y and Y′ together is a single-bonded or adouble-bonded nitrogen bonded to one or more of hydrogen, hydroxyl,alkyl, and trifluoromethyl;

X is selected from hydrogen, alkyl, trifluoromethyl, alkoxy,alkylmercapto, and hydroxyl with the proviso that X is not hydroxyl whenY and Y′ together is oxygen in a compound of formula I wherein R₅ and R₆together is a carbon-carbon bond, or X is NR′R″, where R′ is selectedfrom the group consisting of hydrogen, hydroxyl, alkyl, trifluoromethyl,alkoxy, alkenyl, alkynyl, hydroxyalkyl, polyhydroxyalkyl,alkylaminoalkyl, dialkylaminoalkyl, aminoalkyl, aryl, arylalkyl,arylalkenyl, arylcycloalkyl, arylcycloalkenyl, carbocyclyl, andcarbocycloalkyl where the carbocycle of the carbocyclyl and thecarbocycloalkyl is selected from 7-membered carbocyclic rings containingno double bond, or one, two or three double bonds, 6-memberedcarbocyclic rings containing no double bond, or one or two double bonds,5-membered carbocyclic rings containing no double bond, or one or twodouble bonds, 4-membered carbocyclic rings containing no double bond orone double bond and 3-membered carbocyclic rings containing no doublebond, heterocyclyl, and heterocycloalkyl, where the heterocycle of theheterocyclyl and heterocycloalkyl is selected from 7-memberedheterocyclic rings, 6-membered heterocyclic rings, and 5-memberedheterocyclic rings, and the aryl of the aryl, arylalkyl, arylalkylenyl,arylcycloalkyl, or arylcycloalkenyl structure or the carbocyclic orheterocyclic structure may optionally be substituted with one or more ofhalo, alkyl, trifluoromethyl, hydroxyl, alkoxy, amino, alkylamino,dialkylamino, mercapto, alkylmercapto, carboxamido, aldehydo, cyano,oxo, alkylcarbonyloxy, and sulfonamido; and R″ is selected fromhydrogen, alkyl, hydroxyalkyl, aminoalkyl, dialkylaminoalkyl,cyanoalkyl, haloalkyl, alkylcarbonylalkylcarbonyloxy, pyridyl, and COR₁₁wherein R₁₁ is selected from hydrogen, amino, alkyl, trifluoromethyl,alkoxy, alkylmercapto, and aryl; or R′ and R″ together form a 5-, 6- or7-membered, saturated or unsaturated, heterocyclic ring containing atleast one nitrogen, and optionally oxygen and/or sulfur, and theheterocyclic ring may optionally be substituted with one or more ofhalo, alkyl, trifluoromethyl, hydroxyl, alkoxy, amino, alkylamino,dialkylamino, mercapto, alkylmercapto, carboxamido, aldehydo, cyano,oxo, alkylcarbonyloxy, and sulfonamido; and

E is a substituted or unsubstituted, saturated or unsaturated,7-membered, 6-membered, 5-membered, 4-membered or 3-membered carbocyclicor heterocyclic ring;

wherein at least one compound of formula I or a pharmaceuticallyacceptable salt or prodrug thereof is administered alone or incombination with at least one additional therapeutic agent other than acompound or pharmaceutically acceptable salt or prodrug of formula I.

In one aspect, when R and R₀ are hydrogen, n is 1, three of R₁, R₂, R₃,and R₄ are hydrogens and one is halogen, alkyl, or alkoxy or two of R₁,R₂, R₃ and R₄ are hydrogens and two are alkoxy, R₅ and R₆ together forma carbon-carbon bond, R₇ is hydrogen, R₈ is alkyl, Y and Y′ together isoxygen, X is NR′R″ wherein R″ is hydrogen, and R′ is a substituted aryl,then E cannot be a substituted aryl. For example, when R and R₀ arehydrogen, n is 1, three of R₁, R₂, R₃, and R₄ are hydrogens and one ishalogen, alkyl, or alkoxy or two of R₁, R₂, R₃ and R₄ are hydrogens andtwo are alkoxy, R₅ and R₆ together form a carbon-carbon bond, R₇ ishydrogen, R₈ is alkyl, Y and Y′ together is oxygen, X is NR′R″ whereinR″ is hydrogen, R′ is an aryl substituted with any of halo, alkoxy,amino, alkylamino, dialkylamino and sulfonamido, then E cannot be asubstituted aryl wherein two substituents are identically selected fromhydroxyl and alkoxy.

Examples of conditions treatable by the inhibition of Ras-mediatedbiological processes include, for example, tumor cell growth,proliferation, survival, invasion and metastasis, as well as resistanceto chemotherapy, other molecularly targeted therapeutics, and radiation;and, particularly, a method of therapeutically or prophylacticallytreating conditions caused or exacerbated by hyperactive or mutant Ras,including but not limited to cancer, dysplastic, hyperproliferative orprecancerous conditions of pancreas, colon, lung, skin, breast, head andneck, liver, nervous and accessory cell types of the central andperipheral nervous system.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIG. 1 depicts the chemical structures of sulindac and certainderivatives thereof reportedly having anticancer activity.

FIG. 2 depicts the chemical structures of certain other sulindacderivatives reported to inhibit Ras.

FIG. 3 shows chemical structures of selective Ras-inhibitory compoundsidentified by synthetic lethal screening.

FIG. 4 depicts the results of a Ras Binding Domain (RBD) pulldown assaypaired with Western blot showing the relative levels of Ras activationin a panel of colorectal cancer cell lines.

FIG. 5A reveals a correlation between sensitivity of tumor cells tocompound 004 and Ras activation status of the cells as determined byWestern blot analysis.

FIG. 5B reveals a correlation between sensitivity of tumor cells tocompound 006 and Ras activation status of the cells as determined byWestern blot analysis.

FIG. 5C reveals a correlation between sensitivity of tumor cells tocompound 007 and Ras activation status of the cells as determined byWestern blot analysis.

FIG. 5D reveals a correlation between sensitivity of tumor cells tocompound 010 and Ras activation status of the cells as determined byWestern blot analysis.

FIGS. 6A-6J show Ras-selective tumor cell growth-inhibiting activity ofexemplary Ras-inhibitory compounds 006 (FIG. 6A), 007 (FIG. 6B), 019(FIG. 6C), 029 (FIG. 6D), 002 (FIG. 6E), 010 (FIG. 6F), 011 (FIG. 6G),015 (FIG. 6H), 022 (FIG. 6I) and 035 (FIG. 6J) against human HCT-116colon tumor cells expressing mutant Ras compared to human HT-29 colontumor cells expressing wild-type Ras.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with an embodiment, the present invention provides amethod of inhibiting a human or nonhuman mammalian Ras-mediatedbiological process, which method comprising administering in vivo or invitro a Ras-inhibitory amount of at least one compound of formula I, apharmaceutically acceptable salt or prodrug thereof:

wherein:

R and R₀ are independently selected from hydrogen, hydroxyl, alkyl,trifluoromethyl, amino, alkoxy and alkylamino, or R and R₀ together isoxygen or sulfur, or R and R₀ together is a single-bonded or adouble-bonded nitrogen bonded to one or more of hydrogen, hydroxyl,alkyl, and trifluoromethyl; n is 0, 1 or 2;

R₁, R₂, R₃, and R₄ are independently selected from hydrogen, hydroxyl,halogen, alkyl, trifluoromethyl, alkoxy, and alkylmercapto;

R₅, R₆, R₇, and R₈ are independently selected from hydrogen, alkyl,trifluoromethyl and alkoxy; or R₅ and R₆ together form a carbon-carbonbond;

Y is hydrogen, alkyl, or trifluoromethyl, and Y′ is hydrogen, alkyl,trifluoromethyl, amino, alkylamino, or alkoxy, or Y and Y′ together isoxygen or sulfur, or Y and Y′ together is a single-bonded or adouble-bonded nitrogen bonded to one or more of hydrogen, hydroxyl,alkyl, and trifluoromethyl;

X is selected from hydrogen, alkyl, trifluoromethyl, alkoxy,alkylmercapto, and hydroxyl with the proviso that X is not hydroxyl whenY and Y′ together is oxygen in a compound of formula I wherein R₅ and R₆together is a carbon-carbon bond, or X is NR′R″, where R′ is selectedfrom the group consisting of hydrogen, hydroxyl, alkyl, trifluoromethyl,alkoxy, alkenyl, alkynyl, hydroxyalkyl, polyhydroxyalkyl,alkylaminoalkyl, dialkylaminoalkyl, aminoalkyl, aryl, arylalkyl,arylalkenyl, arylcycloalkyl, arylcycloalkenyl, carbocyclyl, andcarbocycloalkyl where the carbocycle of the carbocyclyl and thecarbocycloalkyl is selected from 7-membered carbocyclic rings containingno double bond, or one, two or three double bonds, 6-memberedcarbocyclic rings containing no double bond, or one or two double bonds,5-membered carbocyclic rings containing no double bond, or one or twodouble bonds, 4-membered carbocyclic rings containing no double bond orone double bond and 3-membered carbocyclic rings containing no doublebond, heterocyclyl, and heterocycloalkyl, where the heterocycle of theheterocyclyl and heterocycloalkyl is selected from 7-memberedheterocyclic rings, 6-membered heterocyclic rings, and 5-memberedheterocyclic rings, and the aryl of the aryl, arylalkyl, arylalkylenyl,arylcycloalkyl, or arylcycloalkenyl structure or the carbocyclic orheterocyclic structure may optionally be substituted with one or more ofhalo, alkyl, trifluoromethyl, hydroxyl, alkoxy, amino, alkylamino,dialkylamino, mercapto, alkylmercapto, carboxamido, aldehydo, cyano,oxo, alkylcarbonyloxy, and sulfonamido; and R″ is selected fromhydrogen, alkyl, hydroxyalkyl, aminoalkyl, dialkylaminoalkyl,cyanoalkyl, haloalkyl, alkylcarbonylalkylcarbonyloxy, pyridyl, and COR₁₁wherein R₁₁ is selected from hydrogen, amino, alkyl, trifluoromethyl,alkoxy, alkylmercapto, and aryl; or R′ and R″ together form a 5-, 6- or7-membered, saturated or unsaturated, heterocyclic ring containing atleast one nitrogen, and optionally oxygen and/or sulfur, and theheterocyclic ring may optionally be substituted with one or more ofhalo, alkyl, trifluoromethyl, hydroxyl, alkoxy, amino, alkylamino,dialkylamino, mercapto, alkylmercapto, carboxamido, aldehydo, cyano,oxo, alkylcarbonyloxy, and sulfonamido; and

E is a substituted or unsubstituted, saturated or unsaturated,7-membered, 6-membered, 5-membered, 4-membered or 3-membered carbocyclicor heterocyclic ring;

wherein at least one compound of formula I or a pharmaceuticallyacceptable salt or prodrug thereof is administered alone or incombination with at least one additional therapeutic agent other than acompound or pharmaceutically acceptable salt or prodrug of formula I.

In an embodiment, E is a carbocyclic or heterocyclic ring, optionallysubstituted with one or more substituents selected from hydroxyl,halogen, alkyl, haloalkyl, cyano, cyanoalkyl, nitro, oxo, alkoxy,formyloxy, amino, alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl,hydroxyalkyl, aldehydo, mercapto, and alkylmercapto, azido, andsubstituted or unsubstituted groups selected from alkylsulfonyl,alkylsulfinyl, alkylsulfinyloxy, alkylsulfonyloxy, alkylcarbonyloxy,carbamate, carbamido, alkoxycarbonyl, alkylaminocarbonyl, aminocarbonyl,sulfonamido, and alkylenedioxy spanning two substituent positions.

In one aspect, when R and R₀ are hydrogen, n is 1, three of R₁, R₂, R₃,and R₄ are hydrogens and one is halogen, alkyl, or alkoxy or two of R₁,R₂, R₃ and R₄ are hydrogens and two are alkoxy, R₅ and R₆ together forma carbon-carbon bond, R₇ is hydrogen, R₈ is alkyl, Y and Y′ together isoxygen, X is NR′R″ wherein R″ is hydrogen, and R′ is a substituted aryl,then E cannot be a substituted aryl. For example, when R and R₀ arehydrogen, n is 1, three of R₁, R₂, R₃, and R₄ are hydrogens and one ishalogen, alkyl, or alkoxy or two of R₁, R₂, R₃ and R₄ are hydrogens andtwo are alkoxy, R₅ and R₆ together form a carbon-carbon bond, R₇ ishydrogen, R₈ is alkyl, Y and Y′ together is oxygen, X is NR′R″ whereinR″ is hydrogen, R′ is an aryl substituted with any of halo, alkoxy,amino, alkylamino, dialkylamino and sulfonamido, then E cannot be asubstituted aryl wherein two substituents are identically selected fromhydroxyl and alkoxy.

The present invention further provides a method of therapeutically orprophylactically treating a human or nonhuman mammalian patient with adisease or condition treatable by inhibition of one or more Ras-mediatedbiological process, which method comprising administering to a patientin need thereof a therapeutically or prophylactically effective amountof at least one ras-inhibitory compound of formula I, a pharmaceuticallyacceptable salt or prodrug thereof:

wherein:

R and R₀ are independently selected from hydrogen, hydroxyl, alkyl,trifluoromethyl, amino, alkoxy and alkylamino, or R and R₀ together isoxygen or sulfur, or R and R₀ together is a single-bonded or adouble-bonded nitrogen bonded to one or more of hydrogen, hydroxyl,alkyl, and trifluoromethyl; n is 0, 1 or 2;

R₁, R₂, R₃, and R₄ are independently selected from hydrogen, hydroxyl,halogen, alkyl, trifluoromethyl, alkoxy, and alkylmercapto;

R₅, R₆, R₇, and R₈ are independently selected from hydrogen, alkyl,trifluoromethyl and alkoxy; or R₅ and R₆ together form a carbon-carbonbond;

Y is hydrogen, alkyl, or trifluoromethyl, and Y′ is hydrogen, alkyl,trifluoromethyl, amino, alkylamino, or alkoxy, or Y and Y′ together isoxygen or sulfur, or Y and Y′ together is a single-bonded or adouble-bonded nitrogen bonded to one or more of hydrogen, hydroxyl,alkyl, and trifluoromethyl;

X is selected from hydrogen, alkyl, trifluoromethyl, alkoxy,alkylmercapto, and hydroxyl with the proviso that X is not hydroxyl whenY and Y′ together is oxygen in a compound of formula I wherein R₅ and R₆together is a carbon-carbon bond, or X is NR′R″, where R′ is selectedfrom the group consisting of hydrogen, hydroxyl, alkyl, trifluoromethyl,alkoxy, alkenyl, alkynyl, hydroxyalkyl, polyhydroxyalkyl,alkylaminoalkyl, dialkylaminoalkyl, aminoalkyl, aryl, arylalkyl,arylalkenyl, arylcycloalkyl, arylcycloalkenyl, carbocyclyl, andcarbocycloalkyl where the carbocycle of the carbocyclyl and thecarbocycloalkyl is selected from 7-membered carbocyclic rings containingno double bond, or one, two or three double bonds, 6-memberedcarbocyclic rings containing no double bond, or one or two double bonds,5-membered carbocyclic rings containing no double bond, or one or twodouble bonds, 4-membered carbocyclic rings containing no double bond orone double bond and 3-membered carbocyclic rings containing no doublebond, heterocyclyl, and heterocycloalkyl, where the heterocycle of theheterocyclyl and heterocycloalkyl is selected from 7-memberedheterocyclic rings, 6-membered heterocyclic rings, and 5-memberedheterocyclic rings, and the aryl of the aryl, arylalkyl, arylalkylenyl,arylcycloalkyl, or arylcycloalkenyl structure or the carbocyclic orheterocyclic structure may optionally be substituted with one or more ofhalo, alkyl, trifluoromethyl, hydroxyl, alkoxy, amino, alkylamino,dialkylamino, mercapto, alkylmercapto, carboxamido, aldehydo, cyano,oxo, alkylcarbonyloxy, and sulfonamido; and R″ is selected fromhydrogen, alkyl, hydroxyalkyl, aminoalkyl, dialkylaminoalkyl,cyanoalkyl, haloalkyl, alkylcarbonylalkylcarbonyloxy, pyridyl, and COR₁₁wherein R₁₁ is selected from hydrogen, amino, alkyl, trifluoromethyl,alkoxy, alkylmercapto, and aryl; or R′ and R″ together form a 5-, 6- or7-membered, saturated or unsaturated, heterocyclic ring containing atleast one nitrogen, and optionally oxygen and/or sulfur, and theheterocyclic ring may optionally be substituted with one or more ofhalo, alkyl, trifluoromethyl, hydroxyl, alkoxy, amino, alkylamino,dialkylamino, mercapto, alkylmercapto, carboxamido, aldehydo, cyano,oxo, alkylcarbonyloxy, and sulfonamido; and

E is a substituted or unsubstituted, saturated or unsaturated,7-membered, 6-membered, 5-membered, 4-membered or 3-membered carbocyclicor heterocyclic ring;

wherein at least one compound of formula I or a pharmaceuticallyacceptable salt or prodrug thereof is administered alone or incombination with at least one additional therapeutic agent other than acompound or pharmaceutically acceptable salt or prodrug of formula I.

In an embodiment, E is a carbocyclic or heterocyclic ring, optionallysubstituted with one or more substituents selected from hydroxyl,halogen, alkyl, haloalkyl, cyano, cyanoalkyl, nitro, oxo, alkoxy,formyloxy, amino, alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl,hydroxyalkyl, aldehydo, mercapto, and alkylmercapto, azido, andsubstituted or unsubstituted groups selected from alkylsulfonyl,alkylsulfinyl, alkylsulfinyloxy, alkylsulfonyloxy, alkylcarbonyloxy,carbamate, carbamido, alkoxycarbonyl, alkylaminocarbonyl, aminocarbonyl,sulfonamido, and alkylenedioxy spanning two substituent positions.

In one aspect, when R and R₀ are hydrogen, n is 1, three of R₁, R₂, R₃,and R₄ are hydrogens and one is halogen, alkyl, or alkoxy or two of R₁,R₂, R₃ and R₄ are hydrogens and two are alkoxy, R₅ and R₆ together forma carbon-carbon bond, R₇ is hydrogen, R₈ is alkyl, Y and Y′ together isoxygen, X is NR′R″ wherein R″ is hydrogen, and R′ is a substituted aryl,then E cannot be a substituted aryl. For example, when R and R₀ arehydrogen, n is 1, three of R₁, R₂, R₃, and R₄ are hydrogens and one ishalogen, alkyl, or alkoxy or two of R₁, R₂, R₃ and R₄ are hydrogens andtwo are alkoxy, R₅ and R₆ together form a carbon-carbon bond, R₇ ishydrogen, R₈ is alkyl, Y and Y′ together is oxygen, X is NR′R″ whereinR″ is hydrogen, R′ is an aryl substituted with any of halo, alkoxy,amino, alkylamino, dialkylamino and sulfonamido, then E cannot be asubstituted aryl wherein two substituents are identically selected fromhydroxyl and alkoxy.

In an embodiment of the above methods, R′, the 7-membered heterocyclicring is selected from azepanyl, oxazepanyl, thiazepanyl, azepinyl,oxepinyl, thiepanyl, homopiperazinyl, diazepinyl and thiazepinyl, the6-membered heterocyclic ring is selected from piperidinyl, oxanyl,thianyl, pyridinyl, pyranyl, thiopyranyl, piperazinyl, morpholinyl,thiomorpholinyl, dioxanyl, dithianyl, diazinyl, oxazinyl, thiazinyl,dioxinyl, dithiinyl, trioxanyl, trithianyl, triazinyl and tetrazinyl,and the 5-membered heterocyclic ring is selected from pyrrolidinyl,tetrahydrofuranyl, tetrahydrothiaphenyl, pyrrolyl, furanyl, thiophenyl,imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl,thiazolidinyl, isothiazolidinyl, dioxolanyl, dithiolanyl, imidazolyl,pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl,furazanyl, oxadiazolyl, thiadiazolyl, dithiazolyl and tetrazolyl.

In an embodiment of the above methods, E is selected from cycloheptanyl,cycloheptenyl, cycloheptadienyl, cycloheptatrienyl, cyclohexanyl,cyclohexenyl, cyclohexadienyl, phenyl, cyclopentanyl, cyclopentenyl,cyclopentadienyl, cyclopropanyl, cyclobutanyl, azepanyl, oxazepanyl,thiazepanyl, azepinyl, oxepinyl, thiepanyl, homopiperazinyl, diazepinyl,thiazepinyl, piperidinyl, oxanyl, thianyl, pyridinyl, pyranyl,thiopyranyl, piperazinyl, morpholinyl, thiomorpholinyl, dioxanyl,dithianyl, diazinyl, oxazinyl, thiazinyl, dioxinyl, dithiinyl,trioxanyl, trithianyl, triazinyl, tetrazinyl, pyrrolidinyl,tetrahydrofuranyl, tetrahydrothiaphenyl, pyrrolyl, furanyl, thiophenyl,imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl,thiazolidinyl, isothiazolidinyl, dioxolanyl, dithiolanyl, imidazolyl,pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl,furazanyl, oxadiazolyl, thiadiazolyl, dithiazolyl, and tetrazolyl, eachof which is substituted or unsubstituted.

In an embodiment of the above methods, E is:

wherein R₁₂, R₁₄, R₁₆, R₁₇, R₁₈ and R₁₉ are independently selected fromhydrogen, hydroxyl, halogen, alkyl, trifluoromethyl, alkoxy, amino,alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl, hydroxyalkyl,aldehydo, mercapto, and alkylmercapto, azido, and substituted orunsubstituted groups selected from alkylsulfonyl, alkylsulfinyl,alkylsulfinyloxy, alkylsulfonyloxy, carbamate, carbamido,alkoxycarbonyl, alkylaminocarbonyl, aminocarbonyl, and sulfonamido.

Examples of compounds utilized in embodiments of the above methodsinclude compounds wherein:

E is a substituted aryl or heteroaryl having at least one alkylsulfinylor alkylsulfonyl substituent on the aryl or heteroaryl ring, or havingat least one alkylmercapto on the aryl ring, or having a p-halo on thearyl ring when R′ is dialkylaminoalkyl; or,

R₅ and R₆ together is a carbon-carbon bond, R″ is hydrogen and R′ is asubstituted arylalkyl, and E is a substituted or unsubstitutedheterocyclic ring selected from the group consisting of pyridinyl,pyrimidinyl, pyrazinyl, imidazolyl, triazinyl, thiophenyl, furanyl,thiazolyl, pyrazolyl and pyrrolyl; or

n is 1 or 2, R₅ and R₆ together form a carbon-carbon bond, Y and Y′ arehydrogens or Y and Y′ together is oxygen, E is a substituted phenyl, Xis hydrogen, alkyl, trifluoromethyl, alkylmercapto or NR′R″ wherein R′is hydrogen, hydroxyl or alkyl, and R″ is hydrogen, alkyl or haloalkyl;or

R₅ and R₆ are independently selected from hydrogen or alkyl or R₅ and R₆together form a carbon-carbon bond, E is a substituted aryl orheteroaryl, X is NR′R″ wherein R′ is hydrogen or hydroxyl and R″ isCOR₁₁ wherein R₁₁ is alkyl, alkoxy or amino; or

R₅, R₆, R₇ and R₈ are independently selected from hydrogen, alkyl andalkoxy, or R₅ and R₆ form a carbon-carbon bond, R₇ is hydrogen, R₈ ishydrogen, alkoxy or alkyl, E is phenyl substituted with at least twohydroxyl groups or at least two alkoxy groups, and Y and Y′ together isoxygen, X is substituted alkoxy or NR′R″, where R′ is selected fromhydrogen, alkyl, alkoxy, alkenyl, alkynyl, hydroxyalkyl,polyhydroxyalkyl, dialkylaminoalkyl, aminoalkyl, arylalkyl, phenyl,indanyl, heterocyclyl, and heterocycloalkyl, where the heterocycle ofthe heterocyclyl and the heterocycloalkyl is selected from pyridinyl,piperidinyl, piperazinyl, pyrrolidinyl, and N-morpholino, and whereinany of the cyclic structures of the R′ may be unsubstituted orsubstituted with one or more of halo, alkoxy, hydroxy, amino,alkylamino, dialkylamino, and sulfonamido; and R″ is selected fromhydrogen, alkyl, alkylamino, cyanoalkyl, haloalkyl, dialkylaminoalkyl,alkylcarbonylalkylcarbonyloxy, and pyridinyl.

In an embodiment of the above methods, wherein the at least one compoundof formula (I) is a compound of formula (II):

wherein:

R and R₀ are independently selected from hydrogen, hydroxyl, alkyl,trifluoromethyl, amino, alkoxy and alkylamino, or R and R₀ together isoxygen or sulfur, or R and R₀ together is a single-bonded or adouble-bonded nitrogen bonded to one or more of hydrogen, hydroxyl,alkyl, and trifluoromethyl; n is 0, 1 or 2;

Y is hydrogen, alkyl, or trifluoromethyl, and Y′ is hydrogen, alkyl,trifluoromethyl, amino, alkylamino, or alkoxy, or Y and Y′ together isoxygen or sulfur, or Y and Y′ together is a single-bonded or adouble-bonded nitrogen bonded to one or more of hydrogen, hydroxyl,alkyl, and trifluoromethyl;

R₁, R₂, R₃, and R₄ are independently selected from hydrogen, hydroxyl,halogen, alkyl, trifluoromethyl, alkoxy, and alkylmercapto;

R₇ and R₈ are independently selected from hydrogen, alkyl,trifluoromethyl and alkoxy;

R₁₂R₁₃, R₁₄, R₁₅, and R₁₆ are independently selected from hydrogen,halogen, alkyl, trifluoromethyl, hydroxyl, alkoxy, formyloxy,alkylcarbonyloxy, hydroxyalkyl, aldehydo, amino, alkylamino, aminoalkyl,alkylaminoalkyl, dialkylamino, mercapto, alkylmercapto, azido, andsubstituted or unsubstituted groups selected from alkylsulfonyl,alkylsulfinyl, alkylsulfinyloxy, alkylsulfonyloxy, carbamate, carbamido,alkoxycarbonyl, alkylaminocarbonyl, aminocarbonyl, and sulfonamido, orany two of R₁₂, R₁₃, R₁₄, R₁₅ and R₁₆ form an alkylenedioxy group;

X is selected from hydrogen, alkyl, trifluoromethyl, alkoxy,alkylmercapto, and hydroxyl with the proviso that X is not hydroxyl whenY and Y′ together is oxygen, or X is NR′R″, where R′ is selected fromthe group consisting of hydrogen, hydroxyl, alkyl, trifluoromethyl,alkoxy, alkenyl, alkynyl, hydroxyalkyl, polyhydroxyalkyl,alkylaminoalkyl, dialkylaminoalkyl, aminoalkyl, arylalkyl, arylalkenyl,arylcycloalkyl, arylcycloalkenyl, aryl, carbocyclyl, and carbocycloalkylwhere the carbocycle of the carbocyclyl and the carbocycloalkyl isselected from 7-membered carbocyclic rings containing no double bond, orone, two or three double bonds, 6-membered carbocyclic rings containingno double bond, or one or two double bonds, 5-membered carbocyclic ringscontaining no double bond, or one or two double bonds, 4-memberedcarbocyclic rings containing no double bond or one double bond and3-membered carbocyclic rings containing no double bond, heterocyclyl,and heterocycloalkyl, where the heterocycle of the heterocyclyl andheterocycloalkyl is selected from furanyl and pyrrolyl, and the aryl ofthe aryl, arylalkyl, arylalkylenyl, arylcycloalkyl, or arylcycloalkenylstructure or the carbocyclic or heterocyclic structure may optionally besubstituted with one or more of halo, alkyl, trifluoromethyl, hydroxyl,alkoxy, amino, alkylamino, dialkylamino, mercapto, alkylmercapto,carboxamido, aldehydo, cyano, oxo, alkylcarbonyloxy, and sulfonamido;and R″ is selected from hydrogen, alkyl, hydroxyalkyl, aminoalkyl,dialkylaminoalkyl, cyanoalkyl, haloalkyl, alkylcarbonylalkylcarbonyloxy,pyridyl, and COR₁₁ wherein R₁₁ is selected from hydrogen, amino, alkyl,trifluoromethyl, alkoxy, alkylmercapto, and aryl; or R′ and R″ togetherform a 5-, 6- or 7-membered, saturated or unsaturated, heterocyclic ringcontaining at least one nitrogen, and optionally oxygen and/or sulfur,and the heterocyclic ring may optionally be substituted with one or moreof halo, alkyl, trifluoromethyl, hydroxyl, alkoxy, amino, alkylamino,dialkylamino, mercapto, alkylmercapto, carboxamido, aldehydo, cyano,oxo, alkylcarbonyloxy, and sulfonamido.

In one aspect, when R and R₀ are hydrogen, n is 1, three of R₁, R₂, R₃,and R₄ are hydrogens and one is halogen, alkyl, or alkoxy or two of R₁,R₂, R₃ and R₄ are hydrogens and two are alkoxy, R₅ and R₆ together forma carbon-carbon bond, R₇ is hydrogen, R₈ is alkyl, Y and Y′ together isoxygen, X is NR′R″ wherein R″ is hydrogen, and R′ is a substituted aryl,then each of R₁₂, R₁₃, R₁₄, R₁₅ and R₁₆ must be hydrogen. For example,when R and R₀ are hydrogen, n is 1, three of R₁, R₂, R₃, and R₄ arehydrogens and one is halogen, alkyl, or alkoxy or two of R₁, R₂, R₃ andR₄ are hydrogens and two are alkoxy, R₅ and R₆ together form acarbon-carbon bond, R₇ is hydrogen, R₈ is alkyl, Y and Y′ together isoxygen, X is NR′R″ wherein R″ is hydrogen, and R′ is an aryl substitutedwith any of halo, alkoxy, amino, alkylamino, dialkylamino andsulfonamido, then no two of R₁₂, R₁₃, R₁₄, R₁₅ and R₁₆ can beidentically selected from hydroxyl and alkoxy.

In another embodiment of the above methods, the at least one compound offormula (I) is a compound of formula (II):

wherein:

R and R₀ are independently selected from hydrogen, hydroxyl, alkyl,trifluoromethyl, amino, alkoxy and alkylamino, or R and R₀ together issulfur, or R and R₀ together is a single-bonded or a double-bondednitrogen bonded to one or more of hydrogen, hydroxyl, alkyl, andtrifluoromethyl; n is 0, 1 or 2;

Y is hydrogen, alkyl, or trifluoromethyl, and Y′ is hydrogen, alkyl,trifluoromethyl, amino, alkylamino, or alkoxy, or Y and Y′ together isoxygen or sulfur, or Y and Y′ together is a single-bonded or adouble-bonded nitrogen bonded to one or more of hydrogen, hydroxyl,alkyl, and trifluoromethyl;

R₁, R₂, R₃ and R₄ are independently selected from hydrogen, hydroxyl,halogen, alkyl, trifluoromethyl, alkoxy, and alkylmercapto;

R₇ and R₈ are independently selected from hydrogen, alkyl,trifluoromethyl and alkoxy;

R₁₂, R₁₃, R₁₄, R₁₅ and R₁₆ are independently selected from hydrogen,halogen, alkyl, trifluoromethyl, hydroxyl, alkoxy, formyloxy,alkylcarbonyloxy, hydroxyalkyl, aldehydo, amino, alkylamino, aminoalkyl,alkylaminoalkyl, dialkylamino, mercapto, alkylmercapto, azido, andsubstituted or unsubstituted groups selected from alkylsulfonyl,alkylsulfinyl, alkylsulfinyloxy, alkylsulfonyloxy, carbamate, carbamido,alkoxycarbonyl, alkylaminocarbonyl, aminocarbonyl, and sulfonamido, orany two of R₁₂, R₁₃, R₁₄, R₁₅ and R₁₆ form an alkylenedioxy group;

X is selected from hydrogen, alkyl, trifluoromethyl, alkoxy,alkylmercapto, and hydroxyl with the proviso that X is not hydroxyl whenY and Y′ together is oxygen, or X is NR′R″, where R′ is selected fromthe group consisting of hydrogen, hydroxyl, alkyl, trifluoromethyl,alkoxy, alkenyl, alkynyl, hydroxyalkyl, polyhydroxyalkyl,alkylaminoalkyl, dialkylaminoalkyl, aminoalkyl, arylalkyl, arylalkenyl,arylcycloalkyl, arylcycloalkenyl, aryl, carbocyclyl, and carbocycloalkylwhere the carbocycle of the carbocyclyl and the carbocycloalkyl isselected from 7-membered carbocyclic rings containing no double bond, orone, two or three double bonds, 6-membered carbocyclic rings containingno double bond, or one or two double bonds, 5-membered carbocyclic ringscontaining no double bond, or one or two double bonds, 4-memberedcarbocyclic rings containing no double bond or one double bond and3-membered carbocyclic rings containing no double bond, heterocyclyl,and heterocycloalkyl, where the heterocycle of the heterocyclyl andheterocycloalkyl is selected from 7-membered heterocyclic rings,6-membered heterocyclic rings, and 5-membered heterocyclic rings, andthe aryl of the aryl, arylalkyl, arylalkylenyl, arylcycloalkyl, orarylcycloalkenyl structure or the carbocyclic or heterocyclic structuremay optionally be substituted with one or more of halo, alkyl,trifluoromethyl, hydroxyl, alkoxy, amino, alkylamino, dialkylamino,mercapto, alkylmercapto, carboxamido, aldehydo, cyano, oxo,alkylcarbonyloxy, and sulfonamido; and R″ is selected from hydrogen,alkyl, hydroxyalkyl, aminoalkyl, dialkylaminoalkyl, cyanoalkyl,haloalkyl, alkylcarbonylalkylcarbonyloxy, pyridyl, and COR₁₁ wherein R₁₁is selected from hydrogen, amino, alkyl, trifluoromethyl, alkoxy,alkylmercapto, and aryl; or R′ and R″ together form a 5-, 6- or7-membered, saturated or unsaturated, heterocyclic ring containing atleast one nitrogen, and optionally oxygen and/or sulfur, and theheterocyclic ring may optionally be substituted with one or more ofhalo, alkyl, trifluoromethyl, hydroxyl, alkoxy, amino, alkylamino,dialkylamino, mercapto, alkylmercapto, carboxamido, aldehydo, cyano,oxo, alkylcarbonyloxy, and sulfonamido.

In one aspect, when R and R₀ are hydrogen, n is 1, three of R₁, R₂, R₃,and R₄ are hydrogens and one is halogen, alkyl, or alkoxy or two of R₁,R₂, R₃ and R₄ are hydrogens and two are alkoxy, R₅ and R₆ together forma carbon-carbon bond, R₇ is hydrogen, R₈ is alkyl, Y and Y′ together isoxygen, X is NR′R″ wherein R″ is hydrogen, and R′ is a substituted aryl,then each of R₁₂, R₁₃, R₁₄, R₁₅ and R₁₆ must be hydrogen. For example,when R and R₀ are hydrogen, n is 1, three of R₁, R₂, R₃, and R₄ arehydrogens and one is halogen, alkyl, or alkoxy or two of R₁, R₂, R₃ andR₄ are hydrogens and two are alkoxy, R₅ and R₆ together form acarbon-carbon bond, R₇ is hydrogen, R₈ is alkyl, Y and Y′ together isoxygen, X is NR′R″ wherein R″ is hydrogen, and R′ is an aryl substitutedwith any of halo, alkoxy, amino, alkylamino, dialkylamino andsulfonamido, then no two of R₁₂, R₁₃, R₁₄, R₁₅ and R₁₆ can beidentically selected from hydroxyl and alkoxy.

In yet another embodiment of the above methods, the at least onecompound of formula (I) is a compound of formula (II):

wherein:

R and R₀ are independently selected from hydrogen, hydroxyl, alkyl,trifluoromethyl, amino, alkoxy and alkylamino, or R and R₀ together isoxygen or sulfur, or R and R₀ together is a single-bonded or adouble-bonded nitrogen bonded to one or more of hydrogen, hydroxyl,alkyl, and trifluoromethyl; n is 0, 1 or 2;

Y is hydrogen, alkyl, or trifluoromethyl, and Y′ is hydrogen, alkyl,trifluoromethyl, amino, alkylamino, or alkoxy, or Y and Y′ together issulfur, or Y and Y′ together is a single-bonded or a double-bondednitrogen bonded to one or more of hydrogen, hydroxyl, alkyl, andtrifluoromethyl;

R₁, R₂, R₃ and R₄ are independently selected from hydrogen, hydroxyl,halogen, alkyl, trifluoromethyl, alkoxy, and alkylmercapto;

R₇ and R₈ are independently selected from hydrogen, alkyl,trifluoromethyl and alkoxy;

R₁₂, R₁₃, R₁₄, R₁₅ and R₁₆ are independently selected from hydrogen,halogen, alkyl, trifluoromethyl, hydroxyl, alkoxy, formyloxy,alkylcarbonyloxy, hydroxyalkyl, aldehydo, amino, alkylamino, aminoalkyl,alkylaminoalkyl, dialkylamino, mercapto, alkylmercapto, azido, andsubstituted or unsubstituted groups selected from alkylsulfonyl,alkylsulfinyl, alkylsulfinyloxy, alkylsulfonyloxy, carbamate, carbamido,alkoxycarbonyl, alkylaminocarbonyl, aminocarbonyl, and sulfonamido, orany two of R₁₂, R₁₃, R₁₄, R₁₅ and R₁₆ form an alkylenedioxy group;

X is selected from hydrogen, alkyl, trifluoromethyl, alkoxy,alkylmercapto, and hydroxyl with the proviso that X is not hydroxyl whenY and Y′ together is oxygen, or X is NR′R″, where R′ is selected fromthe group consisting of hydrogen, hydroxyl, alkyl, trifluoromethyl,alkoxy, alkenyl, alkynyl, hydroxyalkyl, polyhydroxyalkyl,alkylaminoalkyl, dialkylaminoalkyl, aminoalkyl, arylalkyl, arylalkenyl,arylcycloalkyl, arylcycloalkenyl, aryl, carbocyclyl, and carbocycloalkylwhere the carbocycle of the carbocyclyl and the carbocycloalkyl isselected from 7-membered carbocyclic rings containing no double bond, orone, two or three double bonds, 6-membered carbocyclic rings containingno double bond, or one or two double bonds, 5-membered carbocyclic ringscontaining no double bond, or one or two double bonds, 4-memberedcarbocyclic rings containing no double bond or one double bond and3-membered carbocyclic rings containing no double bond, heterocyclyl,and heterocycloalkyl, where the heterocycle of the heterocyclyl andheterocycloalkyl is selected from 7-membered heterocyclic rings,6-membered heterocyclic rings, and 5-membered heterocyclic rings, andthe aryl of the aryl, arylalkyl, arylalkylenyl, arylcycloalkyl, orarylcycloalkenyl structure or the carbocyclic or heterocyclic structuremay optionally be substituted with one or more of halo, alkyl,trifluoromethyl, hydroxyl, alkoxy, amino, alkylamino, dialkylamino,mercapto, alkylmercapto, carboxamido, aldehydo, cyano, oxo,alkylcarbonyloxy, and sulfonamido; and R″ is selected from hydrogen,alkyl, hydroxyalkyl, aminoalkyl, dialkylaminoalkyl, cyanoalkyl,haloalkyl, alkylcarbonylalkylcarbonyloxy, pyridyl, and COR₁₁ wherein R₁₁is selected from hydrogen, amino, alkyl, trifluoromethyl, alkoxy,alkylmercapto, and aryl; or R′ and R″ together form a 5-, 6- or7-membered, saturated or unsaturated, heterocyclic ring containing atleast one nitrogen, and optionally oxygen and/or sulfur, and theheterocyclic ring may optionally be substituted with one or more ofhalo, alkyl, trifluoromethyl, hydroxyl, alkoxy, amino, alkylamino,dialkylamino, mercapto, alkylmercapto, carboxamido, aldehydo, cyano,oxo, alkylcarbonyloxy, and sulfonamido.

In one aspect, when R and R₀ are hydrogen, n is 1, three of R₁, R₂, R₃,and R₄ are hydrogens and one is halogen, alkyl, or alkoxy or two of R₁,R₂, R₃ and R₄ are hydrogens and two are alkoxy, R₅ and R₆ together forma carbon-carbon bond, R₇ is hydrogen, R₈ is alkyl, Y and Y′ together isoxygen, X is NR′R″ wherein R″ is hydrogen, and R′ is a substituted aryl,then each of R₁₂, R₁₃, R₁₄, R₁₅ and R₁₆ must be hydrogen. For example,when R and R₀ are hydrogen, n is 1, three of R₁, R₂, R₃, and R₄ arehydrogens and one is halogen, alkyl, or alkoxy or two of R₁, R₂, R₃ andR₄ are hydrogens and two are alkoxy, R₅ and R₆ together form acarbon-carbon bond, R₇ is hydrogen, R₈ is alkyl, Y and Y′ together isoxygen, X is NR′R″ wherein R″ is hydrogen, and R′ is an aryl substitutedwith any of halo, alkoxy, amino, alkylamino, dialkylamino andsulfonamido, then no two of R₁₂, R₁₃, R₁₄, R₁₅ and R₁₆ can beidentically selected from hydroxyl and alkoxy.

In a further embodiment of the above methods, the at least one compoundof formula (I) is a compound of formula (II):

wherein:

R and R₀ are independently selected from hydrogen, hydroxyl, alkyl,trifluoromethyl, amino, alkoxy and alkylamino, or R and R₀ together isoxygen or sulfur, or R and R₀ together is a single-bonded or adouble-bonded nitrogen bonded to one or more of hydrogen, hydroxyl,alkyl, and trifluoromethyl; n is 0, 1 or 2;

Y is hydrogen, alkyl, or trifluoromethyl, and Y′ is hydrogen, alkyl,trifluoromethyl, amino, alkylamino, or alkoxy, or Y and Y′ together isoxygen or sulfur, or Y and Y′ together is a single-bonded or adouble-bonded nitrogen bonded to one or more of hydrogen, hydroxyl,alkyl, and trifluoromethyl;

R₁, R₂, R₃ and R₄ are independently selected from hydrogen, hydroxyl,halogen, alkyl, trifluoromethyl, alkoxy, and alkylmercapto;

R₇ and R₈ are independently selected from trifluoromethyl and alkoxy;

R₁₂, R₁₃, R₁₄, R₁₅ and R₁₆ are independently selected from hydrogen,halogen, alkyl, trifluoromethyl, hydroxyl, alkoxy, formyloxy,alkylcarbonyloxy, hydroxyalkyl, aldehydo, amino, alkylamino, aminoalkyl,alkylaminoalkyl, dialkylamino, mercapto, alkylmercapto, azido, andsubstituted or unsubstituted groups selected from alkylsulfonyl,alkylsulfinyl, alkylsulfinyloxy, alkylsulfonyloxy, carbamate, carbamido,alkoxycarbonyl, alkylaminocarbonyl, aminocarbonyl, and sulfonamido, orany two of R₁₂, R₁₃, R₁₄, R₁₅ and R₁₆ form an alkylenedioxy group;

X is selected from hydrogen, alkyl, trifluoromethyl, alkoxy,alkylmercapto, and hydroxyl with the proviso that X is not hydroxyl whenY and Y′ together is oxygen, or X is NR′R″, where R′ is selected fromthe group consisting of hydrogen, hydroxyl, alkyl, trifluoromethyl,alkoxy, alkenyl, alkynyl, hydroxyalkyl, polyhydroxyalkyl,alkylaminoalkyl, dialkylaminoalkyl, aminoalkyl, arylalkyl, arylalkenyl,arylcycloalkyl, arylcycloalkenyl, aryl, carbocyclyl, and carbocycloalkylwhere the carbocycle of the carbocyclyl and the carbocycloalkyl isselected from 7-membered carbocyclic rings containing no double bond, orone, two or three double bonds, 6-membered carbocyclic rings containingno double bond, or one or two double bonds, 5-membered carbocyclic ringscontaining no double bond, or one or two double bonds, 4-memberedcarbocyclic rings containing no double bond or one double bond and3-membered carbocyclic rings containing no double bond, heterocyclyl,and heterocycloalkyl, where the heterocycle of the heterocyclyl andheterocycloalkyl is selected from 7-membered heterocyclic rings,6-membered heterocyclic rings, and 5-membered heterocyclic rings, andthe aryl of the aryl, arylalkyl, arylalkylenyl, arylcycloalkyl, orarylcycloalkenyl structure or the carbocyclic or heterocyclic structuremay optionally be substituted with one or more of halo, alkyl,trifluoromethyl, hydroxyl, alkoxy, amino, alkylamino, dialkylamino,mercapto, alkylmercapto, carboxamido, aldehydo, cyano, oxo,alkylcarbonyloxy, and sulfonamido; and R″ is selected from hydrogen,alkyl, hydroxyalkyl, aminoalkyl, dialkylaminoalkyl, cyanoalkyl,haloalkyl, alkylcarbonylalkylcarbonyloxy, pyridyl, and COR₁₁ wherein R₁₁is selected from hydrogen, amino, alkyl, trifluoromethyl, alkoxy,alkylmercapto, and aryl; or R′ and R″ together form a 5-, 6- or7-membered, saturated or unsaturated, heterocyclic ring containing atleast one nitrogen, and optionally oxygen and/or sulfur, and theheterocyclic ring may optionally be substituted with one or more ofhalo, alkyl, trifluoromethyl, hydroxyl, alkoxy, amino, alkylamino,dialkylamino, mercapto, alkylmercapto, carboxamido, aldehydo, cyano,oxo, alkylcarbonyloxy, and sulfonamido.

In one aspect, when R and R₀ are hydrogen, n is 1, three of R₁, R₂, R₃,and R₄ are hydrogens and one is halogen, alkyl, or alkoxy or two of R₁,R₂, R₃ and R₄ are hydrogens and two are alkoxy, R₅ and R₆ together forma carbon-carbon bond, R₇ is hydrogen, R₈ is alkyl, Y and Y′ together isoxygen, X is NR′R″ wherein R″ is hydrogen, and R′ is a substituted aryl,then each of R₁₂, R₁₃, R₁₄, R₁₅ and R₁₆ must be hydrogen. For example,when R and R₀ are hydrogen, n is 1, three of R₁, R₂, R₃, and R₄ arehydrogens and one is halogen, alkyl, or alkoxy or two of R₁, R₂, R₃ andR₄ are hydrogens and two are alkoxy, R₅ and R₆ together form acarbon-carbon bond, R₇ is hydrogen, R₈ is alkyl, Y and Y′ together isoxygen, X is NR′R″ wherein R″ is hydrogen, and R′ is an aryl substitutedwith any of halo, alkoxy, amino, alkylamino, dialkylamino andsulfonamido, then no two of R₁₂, R₁₃, R₁₄, R₁₅ and R₁₆ can beidentically selected from hydroxyl and alkoxy.

In a still further embodiment of the above methods, the at least onecompound of formula (I) is a compound of formula (II):

wherein:

R and R₀ are independently selected from hydrogen, hydroxyl, alkyl,trifluoromethyl, amino, alkoxy and alkylamino, or R and R₀ together isoxygen or sulfur, or R and R₀ together is a single-bonded or adouble-bonded nitrogen bonded to one or more of hydrogen, hydroxyl,alkyl, and trifluoromethyl; n is 0, 1 or 2;

Y is hydrogen, alkyl, or trifluoromethyl, and Y′ is hydrogen, alkyl,trifluoromethyl, amino, alkylamino, or alkoxy, or Y and Y′ together isoxygen or sulfur, or Y and Y′ together is a single-bonded or adouble-bonded nitrogen bonded to one or more of hydrogen, hydroxyl,alkyl, and trifluoromethyl;

R₁, R₂, R₃ and R₄ are independently selected from hydrogen, hydroxyl,halogen, alkyl, trifluoromethyl, alkoxy, and alkylmercapto;

R₇ and R₈ are independently selected from hydrogen, alkyl,trifluoromethyl and alkoxy;

R₁₂, R₁₃, R₁₄, R₁₅ and R₁₆ are independently selected from hydrogen,halogen, alkyl, trifluoromethyl, hydroxyl, alkoxy, formyloxy,alkylcarbonyloxy, hydroxyalkyl, aldehydo, amino, alkylamino, aminoalkyl,alkylaminoalkyl, dialkylamino, mercapto, alkylmercapto, azido, andsubstituted or unsubstituted groups selected from alkylsulfonyl,alkylsulfinyl, alkylsulfinyloxy, alkylsulfonyloxy, carbamate, carbamido,alkoxycarbonyl, alkylaminocarbonyl, aminocarbonyl, and sulfonamido, orany two of R₁₂, R₁₃, R₁₄, R₁₅ and R₁₆ form an alkylenedioxy group;

X is selected from hydrogen, alkyl, trifluoromethyl, alkoxy,alkylmercapto, and hydroxyl with the proviso that X cannot be hydroxylwhen Y and Y′ together is oxygen, or X is NR′R″, where R′ is selectedfrom hydrogen, hydroxyl, alkyl, trifluoromethyl, alkoxy, alkenyl,alkynyl, hydroxyalkyl, polyhydroxyalkyl, alkylaminoalkyl,dialkylaminoalkyl, aminoalkyl, benzyl, phenylalkyl, indanyl, aryl,phenyl, carbocyclyl, and carbocycloalkyl where the carbocycle of thecarbocyclyl and the carbocycloalkyl is selected from 7-memberedcarbocyclic rings containing no double bond, or one, two or three doublebonds, 6-membered carbocyclic rings containing no double bond, or one ortwo double bonds, 5-membered carbocyclic rings containing no doublebond, or one or two double bonds, 4-membered carbocyclic ringscontaining no double bond or one double bond and 3-membered carbocyclicrings containing no double bond, heterocyclyl, and heterocycloalkyl,where the heterocycle of the heterocyclyl and the heterocycloalkyl isselected from azepanyl, oxazepanyl, thiazepanyl, azepinyl, oxepinyl,thiepanyl, homopiperazinyl, diazepinyl, thiazepinyl, oxanyl, thianyl,pyranyl, thiopyranyl, thiomorpholinyl, dioxanyl, dithianyl, diazinyl,oxazinyl, thiazinyl, dioxinyl, dithiinyl, trioxanyl, trithianyl,triazinyl, tetrazinyl, tetrahydrofuranyl, tetrahydrothiaphenyl,pyrrolyl, furanyl, thiophenyl, imidazolidinyl, pyrazolidinyl,oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl,dioxolanyl, dithiolanyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl,thiazolyl, isothiazolyl, triazolyl, furazanyl, oxadiazolyl,thiadiazolyl, dithiazolyl, and tetrazolyl, wherein the carbocyclic orheterocyclic structure may optionally be substituted with one or more ofhalo, alkyl, trifluoromethyl, hydroxyl, alkoxy, amino, alkylamino,dialkylamino, mercapto, alkylmercapto, carboxamido, aldehydo, cyano,oxo, alkylcarbonyloxy, and sulfonamido; R″ is selected from hydrogen,trifluoromethyl, hydroxyalkyl, and COR₁₁ wherein R₁₁ is selected fromthe group consisting of hydrogen, amino, alkyl, trifluoromethyl, alkoxy,alkylmercapto and aryl; or R′ and R″ may be combined to form a 5-, 6- or7-membered ring, saturated or unsaturated, heterocyclic containing atleast one nitrogen, and optionally oxygen and/or sulfur, and theheterocyclic may optionally be substituted with one or more of halo,alkyl, trifluoromethyl, hydroxyl, alkoxy, amino, alkylamino,dialkylamino, mercapto, alkylmercapto, carboxamido, aldehydo, cyano,oxo, alkylcarbonyloxy and sulfonamido.

In one aspect, when R and R₀ are hydrogen, n is 1, three of R₁, R₂, R₃,and R₄ are hydrogens and one is halogen, alkyl, or alkoxy or two of R₁,R₂, R₃ and R₄ are hydrogens and two are alkoxy, R₅ and R₆ together forma carbon-carbon bond, R₇ is hydrogen, R₈ is alkyl, Y and Y′ together isoxygen, X is NR′R″ wherein R″ is hydrogen, and R′ is a substituted aryl,then each of R₁₂, R₁₃, R₁₄, R₁₅ and R₁₆ must be hydrogen. For example,when R and R₀ are hydrogen, n is 1, three of R₁, R₂, R₃, and R₄ arehydrogens and one is halogen, alkyl, or alkoxy or two of R₁, R₂, R₃ andR₄ are hydrogens and two are alkoxy, R₅ and R₆ together form acarbon-carbon bond, R₇ is hydrogen, R₈ is alkyl, Y and Y′ together isoxygen, X is NR′R″ wherein R″ is hydrogen, and R′ is an aryl substitutedwith any of halo, alkoxy, amino, alkylamino, dialkylamino andsulfonamido, then no two of R₁₂, R₁₃, R₁₄, R₁₅ and R₁₆ can beidentically selected from hydroxyl and alkoxy.

In an embodiment of the method of inhibiting a Ras-mediated biologicalprocess with a compound of formula (I), X is NR′R″ where R′ is selectedfrom alkyl, trifluoromethyl, alkenyl, alkynyl, hydroxyalkyl,alkylaminoalkyl, dialkylaminoalkyl, arylalkyl selected from the groupconsisting of benzyl, phenylalkyl, indanyl, heterocyclyl, andheterocycloalkyl, where the heterocycle is selected from furanyl,pyrrolyl, thiophenyl, and imidazolyl, and the cyclic structure ofheterocyclyl and heterocycloalkyl is optionally substituted with one ormore of halo, alkyl, trifluoromethyl, hydroxy, alkoxy, amino,alkylamino, dialkylamino, mercapto, alkylmercapto, and carboxamido; R″is selected from hydrogen, alkyl, trifluoromethyl, cyanoalkyl, anddialkylaminoalkyl, or R′ and R″ together form a 5, 6, or 7-memberheterocyclic ring, saturated or unsaturated, substituted orunsubstituted, that contains at least one nitrogen and optionallyoxygen.

In a particular embodiment of the above method, X is NR′R″ where R′ isselected from alkylaminoalkyl, dialkylaminoalkyl, arylalkyl, benzyl,heterocyclyl, and heterocycloalkyl where the heterocycle is selectedfrom furanyl, pyrrolyl, and thiophenyl, and the cyclic structure ofheterocyclyl and heterocycloalkyl is optionally substituted with one ormore of halo, alkyl, trifluoromethyl, hydroxy, alkoxy, amino,alkylamino, and dialkylamino; R″ is selected from hydrogen, alkyl,trifluoromethyl or dialkylaminoalkyl, or R′ and R″ together form a 5, 6,or 7-member heterocyclic ring, saturated or unsaturated, substituted orunsubstituted, that contains at least one nitrogen and optionallyoxygen.

In a more particular embodiment of the above method, X is NR′R″ where R′is selected from dialkylaminoalkyl, arylalkyl, benzyl, heterocyclyl, andheterocycloalkyl where the heterocycle is selected from furanyl andpyrrolyl, and the cyclic structure may optionally be substituted withone or more of halo, alkyl, trifluoromethyl, alkoxy, alkylamino anddialkylamino; and R″ is selected from hydrogen, alkyl, trifluoromethylor dialkylaminoalkyl.

In a further particular embodiment of the above method, X is NR′R″ whereR′ is benzyl, or a heterocyclyl or heterocycloalkyl selected from2-furfuryl, 2-pyrrolylmethyl, and (1-methyl-1H-pyrrol-2-yl)methyl; andR″ is hydrogen.

In an example of the embodiment of the above method, X is NR′R″ where R′is heterocyclyl or heterocycloalkyl selected 2-furfuryl,(1H-pyrrol-2-yl)methyl, and (1-methyl-1H-pyrrol-2-yl)methyl; and R″ ishydrogen.

In any of the above embodiments, R and R₀ are independently selectedfrom hydrogen and hydroxyl, and R₁, R₂, R₃ and R₄ are independentlyselected from halogen, alkoxy, alkyl and trifluoromethyl; n is 1; R₁₂,R₁₃, R₁₄, R₁₅ and R₁₆ are independently selected from hydrogen, halogen,alkyl, trifluoromethyl, hydroxyl, alkoxy, formyloxy, alkylcarbonyloxy,hydroxyalkyl, aldehydo, amino, alkylamino, aminoalkyl, alkylaminoalkyl,dialkylamino, mercapto, alkylmercapto, azido, and substituted orunsubstituted groups selected from alkyl sulfonyl, alkylsulfinyl,alkylsulfinyloxy, alkylsulfonyloxy, carbamate, carbamido, andsulfonamido, or any two of R₁₂, R₁₃, R₁₄, R₁₅ and R₁₆ form analkylenedioxy group.

For example, R₁, R₂, R₃ and R₄ are independently selected from halogen,alkoxy, alkyl and trifluoromethyl; three of R₁₂, R₁₃, R₁₄, R₁₅ and R₁₆are independently selected from hydrogen, halogen, alkyl,trifluoromethyl, hydroxyl, alkoxy, formyloxy, alkylcarbonyloxy,hydroxyalkyl, aldehydo, amino, alkylamino, aminoalkyl, alkylaminoalkyl,dialkylamino, mercapto, alkylmercapto, azido, and substituted orunsubstituted groups selected from alkyl sulfonyl, alkylsulfinyl,alkylsulfinyloxy, alkylsulfonyloxy, carbamate, carbamido, andsulfonamido, and one of R₁₂, R₁₃, R₁₄, R₁₅ and R₁₆ is independentlyselected from hydroxyl, hydroxyalkyl, amino, alkylamino, dialkylamino,mercapto, and alkylmercapto.

In the above embodiment, three of R₁₂, R₁₃, R₁₄, R₁₅ and R₁₆ areindependently selected from hydrogen, halogen, alkyl, trifluoromethyl,alkoxy, amino, alkylamino, aminoalkyl, alkylaminoalkyl, dialkylamino,mercapto, and alkylmercapto, and one of R₁₂, R₁₃, R₁₄, R₁₅ and R₁₆ isindependently selected from hydroxyl, hydroxyalkyl, aldehydo, amino,alkylamino, dialkylamino, mercapto, and alkylmercapto; and R₈ is methyl.

In an embodiment of the methods of the invention involving a compound offormula (II), R₂ is selected from halogen, alkoxy and alkylmercapto, R₁and R₃ are hydrogen; and three of R₁₂, R₁₃, R₁₄, R₁₅ and R₁₆ areindependently selected from hydrogen, halogen, alkyl, trifluoromethyl,alkoxy, alkylamino, alkylaminoalkyl, dialkylamino, and alkylmercapto,and one of R₁₂, R₁₃, R₁₄, R₁₅ and R₁₆ is independently selected fromhydroxyl, hydroxyalkyl, amino, alkylamino, dialkylamino, mercapto, andalkylmercapto.

In a particular embodiment of the above methods, R₂ is selected fromhalogen and alkoxy, and R₁ and R₃ are hydrogen, more particularly R₂ isselected from fluoro and methoxy.

In accordance with embodiments of the methods, the compound employedtherein is selected from:

-   (Z)—N-(furan-2-ylmethyl)-2-(1-(4-hydroxy-3,5-dimethylbenzylidene)-5-methoxy-2-methyl-1H-inden-3-yl)acetamide    (001),-   (Z)-2-(5-fluoro-1-(4-formoxy-3,5-dimethoxybenzylidene)-2-methyl-1H-inden-3-yl)-N-(furan-2-ylmethyl)acetamide    (002),-   (Z)—N-(2-(dimethylamino)ethyl)-2-(1-(4-hydroxy-3,5-dimethoxybenzylidene)-5-methoxy-2-methyl-1H-inden-3-yl)acetamide    (003),-   (Z)-2-(1-(4-hydroxy-3,5-dimethoxybenzylidene)-5-methoxy-2-methyl-1H-inden-3-yl)-N-(pyridin-3-yl)acetamide    (004),-   (Z)-2-(1-(4-hydroxy-3,5-dimethoxybenzylidene)-5-methoxy-2-methyl-1H-inden-3-yl)-N-(1-methylpyrrolidin-3-yl)acetamide    (005),-   (Z)—N-(furan-2-ylmethyl)-2-(1-(4-hydroxy-3,5-dimethoxybenzylidene)-5-methoxy-2-methyl-1H-inden-3-yl)acetamide    (006),-   (Z)-2-(5-fluoro-1-(4-hydroxy-3,5-dimethoxybenzylidene)-2-methyl-1H-inden-3-yl)-N-(furan-2-ylmethyl)acetamide    (007),-   (Z)—N-(furan-2-ylmethyl)-2-(1-(4-hydroxy-3,5-dimethoxybenzylidene)-5,6-dimethoxy-2-methyl-1H-inden-3-yl)acetamide    (008),-   (Z)-2-(1-(4-hydroxy-3,5-dimethoxybenzylidene)-5-methoxy-2-methyl-1H-inden-3-yl)-N-(pyridin-3-ylmethyl)acetamide    (009),-   (Z)-2-(5-fluoro-1-(4-hydroxy-3,5-dimethoxybenzylidene)-2-methyl-1H-inden-3-yl)-N-(pyridin-2-ylmethyl)acetamide    (010),-   (Z)-2-(5-fluoro-1-(4-hydroxy-3,5-dimethoxybenzylidene)-2-methyl-1H-inden-3-yl)-N-(pyridin-3-ylmethyl)acetamide    (011),-   (Z)—N-(furan-2-ylmethyl)-2-(1-(4-hydroxy-3-methoxybenzylidene)-5-methoxy-2-methyl-1H-inden-3-yl)acetamide    (012),-   (Z)-2-(1-(3-bromo-4-hydroxy-5-methoxybenzylidene)-5-methoxy-2-methyl-1H-inden-3-yl)-N-(furan-2-ylmethyl)acetamide    (013),-   (Z)-2-(1-(3-chloro-4-hydroxy-5-methoxybenzylidene)-5-methoxy-2-methyl-1H-inden-3-yl)-N-(furan-2-ylmethyl)acetamide    (014),-   (Z)—N-benzyl-2-(5-fluoro-1-(4-hydroxy-3,5-dimethoxybenzylidene)-2-methyl-1H-inden-3-yl)acetamide    (015),-   (Z)-2-(1-(3-fluoro-4-hydroxy-5-methoxybenzylidene)-5-methoxy-2-methyl-1H-inden-3-yl)-N-(furan-2-ylmethyl)acetamide    (016),-   (Z)-2-(5-fluoro-1-((7-hydroxybenzo[d][1,3]dioxol-5-yl)methylene)-2-methyl-1H-inden-3-yl)-N-(furan-2-ylmethyl)acetamide    (017),-   (Z)—N-((1H-pyrrol-2-yl)methyl)-2-(5-fluoro-1-(4-hydroxy-3,5-dimethoxybenzylidene)-2-methyl-1H-inden-3-yl)acetamide    (018),-   (Z)-2-(5-fluoro-1-(4-hydroxy-3,5-dimethoxybenzylidene)-2-methyl-1H-inden-3-yl)-N-((1-methyl-1H-pyrrol-2-yl)methyl)acetamide    (019),-   (Z)—N-(furan-2-ylmethyl)-2-(1-(3-hydroxy-4-methoxybenzylidene)-5-methoxy-2-methyl-1H-inden-3-yl)acetamide    (020),-   (Z)-2-(5-fluoro-1-(4-(hydroxymethyl)-3,5-dimethoxybenzylidene)-2-methyl-1H-inden-3-yl)-N-(furan-2-ylmethyl)acetamide    (021),-   (Z)—N-((1H-pyrrol-2-yl)methyl)-2-(1-(4-hydroxy-3,5-dimethoxybenzylidene)-5-methoxy-2-methyl-1H-inden-3-yl)acetamide    (022),-   (Z)-2-(1-(4-hydroxy-3,5-dimethoxybenzylidene)-5-methoxy-2-methyl-1H-inden-3-yl)-N-(pyridin-2-ylmethyl)acetamide    (028),-   (Z)-2-(5-fluoro-1-(4-mesyloxy-3,5-dimethoxybenzylidene)-2-methyl-1H-inden-3-yl)-N-(furan-2-ylmethyl)acetamide    (029),-   (Z)-2-(1-(4-hydroxy-3,5-dimethoxybenzylidene)-5-methoxy-2-methyl-1H-inden-3-yl)-N-phenylacetamide    (034),-   (Z)-2-(5-fluoro-1-(4-hydroxy-3,5-dimethoxybenzylidene)-2-methyl-1H-inden-3-yl)-N-phenylacetamide    (035),-   (Z)-2-(1-(4-aminocarbonyl-3,5-dimethoxybenzylidene)-5-fluoro-2-methyl-1H-inden-3-yl)-N-((1-methyl-1H-pyrrol-2-yl)methyl)acetamide    (065),-   (Z)-2-(1-(3,5-dimethoxy-4-sulfamoylbenzylidene)-5-methoxy-2-methyl-1H-inden-3-yl)-N-(1-methylpyrrolidin-3-yl)acetamide    (067),-   (Z)-2-(1-(3,5-dimethoxy-4-sulfamoylbenzylidene)-5-fluoro-2-methyl-1H-inden-3-yl)-N-(pyridin-2-ylmethyl)acetamide    (068), and-   (Z)-2-(1-(3,5-dimethoxy-4-ureidobenzylidene)-5-fluoro-2-methyl-1H-inden-3-yl)-N-((4-methylpyridin-3-yl)methyl)acetamide    (069),

or the corresponding Z- or E-isomer thereof, prodrug, or salt thereof

The structural formulas of the above compounds are as follows:

In accordance with an embodiment, the disease or condition treatable bythe inhibition of one or more Ras-mediated biological process is cancer,neurofibromatosis, or Costello syndrome, particularly cancer.

In accordance with an embodiment, the Ras-mediated biological process isselected from growth, proliferation, survival, metastasis, drugresistance and radiation resistance of a tumor cell.

Examples of cancers include pancreatic cancer, lung cancer, colorectalcancer, melanoma, ovarian cancer, renal cancer, prostate cancer, headand neck cancer, endocrine cancer, uterine cancer, breast cancer,sarcoma cancer, gastric cancer, hepatic cancer, esophageal cancer,central nervous system cancer, brain cancer, hepatic cancer, germlinecancer, lymphoma, and leukemia, particularly pancreatic cancer,colorectal cancer, and lung cancer.

In an embodiment, the cancer is drug-resistant or radiation-resistant.

The invention further provides a method wherein the patient ispre-selected by utilizing an assay of the patient's tissue, blood ortumor for an abnormal, mutant or hyperactive ras gene or Ras protein, oran aberrant Ras-mediated biological process. Thus, for example, thepatient's tissue, blood or tumor contains an abnormal, mutant orhyperactive ras gene or Ras protein, or aberrant Ras-mediated biologicalprocess.

In a preferred embodiment, a Ras-inhibitory compound can be identifiedfrom one or more compounds of formulas I—II by an assay of Rasinhibition. Some representative assays of selective Ras inhibition areillustrated in the examples that follow herein. As used herein, theterminology selective “Ras inhibition” means selective, preferential orspecific inhibition of aberrant Ras-mediated cellular processes, suchas, for example, accelerated or aberrant cell growth, proliferation,survival, and invasiveness, relative to these processes in cells ortissues with normal or non-aberrant Ras and Ras-mediated processes.

Experimentally, selective Ras inhibition can be shown, for example, bydetermining the ratio (numerator/denominator) of a given compound'spotency (e.g., IC₅₀) to inhibit the growth of cells with “normal” or“wild-type” Ras (numerator) relative to that of cells with mutatedand/or activated Ras (denominator). The terminology used herein for suchan experimentally determined ratio is “selectivity” or “selectivityindex”, which may be further denoted by showing the respective celltypes used to determine the numerical ratio (e.g., HT-29/A549;Caco-2/SW-480; HT-29/SW-480; HT-29/CCT-116). For a given compound, a“selectivity” value or “selectivity index” of greater than 1 (one),preferably greater than 10 (ten), more preferably greater than 100 (onehundred) and even more preferably greater than 1000 (one thousand)indicates said compound selectively inhibits hyperactive Ras and/orRas-mediated cellular functions, such as those which may drive oraccelerate cancer cell growth, proliferation, metastasis, resistance todrugs or radiation, and the like. Conversely, compounds with selectivityindex less than or equal to one (<1) are, by definition herein,“non-selective” with respect to Ras.

The present invention further provides a pharmaceutical compositioncomprising a compound as described above, a pharmaceutically acceptablesalt or prodrug thereof, and a pharmaceutically acceptable carrier.

In accordance with an embodiment, the pharmaceutical composition mayfurther include at least one additional therapeutic agent other than acompound of formula I-II.

In a more preferred embodiment of the present invention, theaforementioned assay of Ras inhibition employs one or more isogenic cellline pair(s), wherein both of the lines share the same geneticbackground except that one of the lines (“mutant line”) contains one ormore mutated or hyperactive ras gene(s), Ras protein(s) and/or aberrantRas-mediated biological process(es), and the other line (“normal line”)lacks such mutation(s) or aberrant function(s).

In a further preferred embodiment of the present invention, theaforementioned assay employing isogenic cell line(s) enables thedetermination and calculation of a Ras-Inhibitory Specificity Index(RISI). One experimental approach to determination of such a RISI may,for example, comprise determining the ratio of the producing a specifiedeffect on the normal line, such as, for example, 50% growth inhibitionin a specified period of time, divided by the concentration of the samecompound producing the same specified effect (e.g., 50% growthinhibition in the same specified period of time) on the mutant line.

Whereas in the aforementioned approach, the 50% growth inhibition valuesmay be obtained by testing the compound against both normal and mutantcell lines at multiple concentrations over a specified concentrationrange, for example 10 nM-10,000 nM, an alternate, more streamlinedapproach to determining a RISI value could comprise measuring the ratioof percentage growth inhibition in a given period of time by a specifiedsingle concentration of the compound, for example 250 nM, selected fromwithin a range of concentrations, for example from within a range of 10nM-10,000 nM, against the mutant (numerator) relative to the normal cellline (denominator). This approach may be generally more applicable tolarger-scale or preliminary screening of groups of individual compoundsor mixtures thereof to obtain a preliminary or screening RISI, whereas aRISI determined using concentration ranges to determine 50% growthinhibition values may be more precise. A RISI value obtained for a givencompound by either approach may be less than, equal to or greater than 1(one), and a RISI value of greater than 1 (one) indicates said compoundselectively inhibits Ras or Ras-mediated cellular functions.

In a highly preferred embodiment of the present invention, the employedassay of Ras inhibition enables identification of a compound from one ormore compounds of formula I or II having a RISI of greater than 1,preferably greater than 10, more preferably greater than 100, and evenmore preferably greater than 1000.

The present invention yet further provides a pharmaceutical compositioncomprising a therapeutically effective amount of Ras-inhibitory activityfrom one or more Ras-inhibitory compound(s) of formula I or II, orpharmaceutically acceptable salt(s) or prodrug(s) thereof, alone or incombination with at least one additional therapeutic agent. Thetherapeutically effective amount can be that amount provided by aRas-inhibiting and/or a disease-process inhibiting effective amount,such as an anticancer effective amount, of a compound of formula I orII.

In addition, the present invention provides a method of therapeuticallyor prophylactically treating a condition treatable by the inhibition ofRas-mediated biological processes including, for example, tumor cellgrowth, proliferation, survival, invasion and metastasis, as well asresistance to chemotherapy, other molecularly targeted therapeutics, andradiation; and, a method of therapeutically or prophylactically treatingcancers harboring hyperactive or mutant Ras. These methods compriseadministering a therapeutically or prophylactically effective amount ofRas-inhibiting activity from at least one Ras-inhibitory compound, orpharmaceutically acceptable salt or prodrug thereof, of formula I or II.

For example, the disease or condition treatable by the inhibition of oneor more Ras-mediated biological process is cancer, neurofibromatosis, orCostello syndrome. In an embodiment, the Ras-mediated biological processis selected from growth, proliferation, survival, metastasis, drugresistance and radiation resistance of a tumor cell.

In an embodiment, the cancer is selected from pancreatic cancer, lungcancer, colorectal cancer, melanoma, ovarian cancer, renal cancer,prostate cancer, head and neck cancer, endocrine cancer, uterine cancer,breast cancer, sarcoma cancer, gastric cancer, hepatic cancer,esophageal cancer, central nervous system cancer, brain cancer, hepaticcancer, germline cancer, lymphoma, and leukemia, particularly pancreaticcancer, colorectal cancer, and lung cancer. In accordance with anembodiment, the cancer is drug-resistant or radiation-resistant.

In an embodiment of the above method, the patient is pre-selected byutilizing an assay of the patient's tissue, blood or tumor for anabnormal, mutant or hyperactive ras gene or Ras protein, or an aberrantRas-mediated biological process.

In an embodiment, the patient's tissue, blood or tumor contains anabnormal, mutant or hyperactive ras gene or Ras protein, or aberrantRas-mediated biological process.

The compounds in the present invention also can be in the form of apharmaceutically acceptable salt, which may include, for example, thesalt of one or more acidic substituents (e.g. a carboxylic salt, asulfonic acid salt, and the like) and the salt of one or more basicsubstituents (e.g. the salt of an amine, and the like). Suitable saltsof acidic substituents include, for example, metal salts (e.g. sodiumsalts, potassium salts, magnesium salts, zinc salts, and the like) andammonium salts (e.g., NH₄+ salts, alkylammonium salts, quaternaryammonium salts, and the like). Suitable salts of basic substituentsinclude, for example, acid addition salts (e.g., hydrochloride salts,hydrobromide salts, carboxylate salts (e.g., acetate salts), sulfatesalts, sulfonate salts (e.g., mesylate salts), phosphate salts,quaternary ammonium salts, and the like.

Compounds employed in the present invention can also be provided as aprodrug, which is a drug derivative or drug precursor compound thattypically is inactive or less than fully active until it is converted inthe body through a normal metabolic process such as, for example,hydrolysis of an ester or amide form of the drug, to the active drug. Aprodrug may be selected and used instead of the parent drug because, forexample, in its prodrug form it is less toxic, and/or may have betterabsorption, distribution, metabolism and excretion (ADME)characteristics, and the like, than the parent drug. A prodrug mightalso be used to improve how selectively the drug interacts with cells orprocesses that are not its intended target. This approach may beemployed particularly, for example, to prevent or decrease adverseeffects, especially in cancer treatments, which may be especially proneto having severe unintended and undesirable side effects.

The term “prodrug” denotes a derivative of a compound, which derivative,when administered to warm-blooded animals, e.g. humans, is convertedinto the compound (drug). For example, the enzymatic and/or chemicalhydrolytic cleavage of a derivative compound of the present inventionoccurs in such a manner that the proven drug form is released, and themoiety or moieties split off remain nontoxic or are metabolized so thatnontoxic metabolites are produced. For example, a carboxylic acid groupcan be esterified, e.g., with a methyl group or ethyl group to yield anester. When an ester is administered to a subject, the ester is cleaved,enzymatically or non-enzymatically, reductively, oxidatively, orhydrolytically, to reveal the anionic group. An anionic group can beesterified with moieties (e.g., acyloxymethyl esters) which are cleavedto reveal an intermediate compound which subsequently decomposes toyield the active compound.

The prodrugs can be prepared in situ during the isolation andpurification of the compounds, or by separately reacting the purifiedcompound with a suitable derivatizing agent. For example, hydroxy groupscan be converted into esters via treatment with a carboxylic acid in thepresence of a catalyst. Examples of cleavable alcohol prodrug moietiesinclude substituted or unsubstituted, branched or unbranched alkyl estermoieties, e.g., ethyl esters, alkenyl esters, di-alkylamino alkylesters, e.g., dimethylaminoethyl ester, acylamino alkyl esters, acyloxyalkyl esters (e.g., pivaloyloxymethyl ester), aryl esters, e.g., phenylester, aryl-alkyl esters, e.g., benzyl ester, optionally substituted,e.g., with methyl, halo, or methoxy substituents aryl and aryl-alkylesters, amides, alkyl amides, di-alkyl amides, and hydroxy amides.

Knowing the disclosures herein, it will be appreciated also that acompound of the present invention can be in the form of a prodrug, andthat such prodrugs can be prepared using reagents and synthetictransformations that are well-known to those having ordinary skill inthe art. The effectiveness of a particular prodrug can be determinedusing one or more analytical methods (e.g. pharmacokinetics, bioassays,in vivo efficacy studies, and the like) that are well-known to those ofordinary skill in the art.

More specifically, a prodrug of a compound of formula I or II may beprepared using routine chemical procedures, such as the exemplaryprocedures described herein. For example, any of the E groups can besubstituted on the ring with a group of the formula Q-U—, for example,

wherein U is selected from the group consisting of oxygen, sulfur,nitrogen, OCH₂, SCH₂ and NHCH₂; and Q is selected from the groupconsisting of PEG-CO, HCO, acetyl, amino acid, substituted benzoic acidand phosphoric acid.

Suitable prodrugs may include, but not be limited to, those illustratedbelow for a compound of formula IV, specifically as exemplary prodrugderivatives of compound 015:

wherein U is selected from the group consisting of oxygen, sulfur,nitrogen, OCH₂, SCH₂ and NHCH₂; and Q is selected from the groupconsisting of PEG-CO, HCO, acetyl, amino acid, substituted benzoic acidand phosphoric acid.

More specific examples are depicted below for compounds 007, 010 and011, respectively:

As used herein, the “alkyl” part of any of the substituents describedherein, e.g., but not limited to, alkyl, alkylamino, alkylmercapto,hydroxyalkyl, polyhydroxyalkyl, alkylaminoalkyl, aminoalkyl, arylalkyl,arylcycloalkyl, heterocycloalkyl, arylalkylenyl, arylcycloalkyl,dialkylamino, alkylcarbonyloxy, dialkylaminoalkyl, cyanoalkyl,haloalkyl, alkylcarbonylalkylcarbonyloxy, dialkylalkylaminoalkyl,alkylsulfonyl, alkylsulfinyl, alkylsulfinyloxy, alkylsulfonyloxy,alkylenedioxy, carbocycloalkyl, and phenylalkyl, means a straight-chainor branched-chain saturated alkyl which can contain from 1-20 carbonatoms, for example from 1 to about 10 carbon atoms, or from 1 to about 8carbon atoms, or, preferably, lower alkyl, i.e., from 1 to 6 carbonatoms.

Examples of alkyls include methyl, ethyl, propyl, isopropyl, n-butyl,sec-butyl, isobutyl, tert-butyl, pentyl, isoamyl, hexyl, octyl,dodecanyl, octadecyl, and the like. Alkyl substituents can beunsubstituted or substituted, for example with at least one substituentselected from the group consisting of a halogen, a nitro, an amino, ahydroxyl, a thio, an acyl, a mercapto, and a cyano.

The term “alkenyl” means a straight-chain or branched-chain alkenylhaving one or more double bonds. Unless otherwise specified, the alkenylcan contain from 2 to about 10 carbon atoms, for example from 2 to about8 carbon atoms, or preferably from 2 to about 6 carbon atoms. Examplesof alkenyls include vinyl, allyl, 1,4-butadienyl, and isopropenylsubstituents, and the like.

The term “alkynyl” means a straight-chain or branched-chain alkynylhaving one or more triple bonds. Unless otherwise specified, alkynylscan contain from 2 to about 10 carbon atoms, for example, from 2 toabout 8 carbon atoms, or preferably, from 2 to about 6 carbon atoms.Examples of alkynyls include ethynyl, propynyl (propargyl), butynyl, andthe like. Alkenyl, alkynyl substituents can be unsubstituted orsubstituted, for example, with at least one substituent selected fromthe group consisting of a halogen, a nitro, an amino, a hydroxyl, athio, an acyl, an alkyl, and a cyano.

The term “aryl” means an aromatic carbocyclic radical, as commonlyunderstood in the art, and includes monocyclic and polycyclic aromaticssuch as, for example, phenyl and naphthyl rings. Preferably, the arylcomprises one or more six-membered rings including, for example, phenyl,naphthyl, biphenyl, and the like. Typically, the aryl comprises six ormore carbon atoms in the ring skeleton thereof (e.g., from 6 to about 10carbon atoms making up the ring). Unless specified otherwise, “aryl” byitself refers to unsubstituted aryl groups and does not coversubstituted aryl groups. Substituted aryl can be an aryl substituted,for example, with at least one substituent selected from the groupconsisting of a halogen, a nitro, an amino, a hydroxyl, a thio, an acyl,and alkyl, and a cyano. It is to be noted that arylalkyl, benzyl, orheteroaryl groups are not considered “aryl” in accordance with thepresent invention.

In accordance with the invention, the term “heteroaryl” refers to acyclic aromatic radical having from five to ten ring atoms of which atleast one atom is O, S, or N, and the remaining atoms are carbon.Examples of heteroaryl radicals include pyridyl, pyrazinyl, pyrimidinyl,pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl,thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, andisoquinolinyl.

Whenever a range of the number of atoms in a structure is indicated(e.g., a C₁₋₁₂, C₁₋₈, C₁₋₆, or C₁₋₄ alkyl, alkylamino, etc.), it isspecifically contemplated that any sub-range or individual number ofcarbon atoms falling within the indicated range also can be used. Thus,for instance, the recitation of a range of 1-8 carbon atoms (e.g.,C₁-C₈), 1-6 carbon atoms (e.g., C₁-C₆), 1-4 carbon atoms (e.g., C₁-C₄),1-3 carbon atoms (e.g., C₁-C₃), or 2-8 carbon atoms (e.g., C₂-C₈) asused with respect to any chemical group (e.g., alkyl, alkylamino, etc.)referenced herein encompasses and specifically describes 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, and/or 12 carbon atoms, as appropriate, as well asany sub-range thereof (e.g., 1-2 carbon atoms, 1-3 carbon atoms, 1-4carbon atoms, 1-5 carbon atoms, 1-6 carbon atoms, 1-7 carbon atoms, 1-8carbon atoms, 1-9 carbon atoms, 1-10 carbon atoms, 1-11 carbon atoms,1-12 carbon atoms, 2-3 carbon atoms, 2-4 carbon atoms, 2-5 carbon atoms,2-6 carbon atoms, 2-7 carbon atoms, 2-8 carbon atoms, 2-9 carbon atoms,2-10 carbon atoms, 2-11 carbon atoms, 2-12 carbon atoms, 3-4 carbonatoms, 3-5 carbon atoms, 3-6 carbon atoms, 3-7 carbon atoms, 3-8 carbonatoms, 3-9 carbon atoms, 3-10 carbon atoms, 3-11 carbon atoms, 3-12carbon atoms, 4-5 carbon atoms, 4-6 carbon atoms, 4-7 carbon atoms, 4-8carbon atoms, 4-9 carbon atoms, 4-10 carbon atoms, 4-11 carbon atoms,and/or 4-12 carbon atoms, etc., as appropriate).

In light of the disclosures of the present invention, it will beappreciated that the compounds used in the present invention can beobtained by methods known to those of ordinary skill in the art, forexample, by structurally modifying a given compound or by directsynthesis from available precursors using routine synthetictransformations that are well-known in the art. For example, a compoundof formula I or II can be synthesized according to the general approachdepicted in Scheme I:

Specific illustrations using the general synthetic approach are providedin the examples that follow herein. Furthermore, one skilled in the artand knowing the disclosures of the present invention will appreciatethat any of compounds of formula I or II can be synthesized usingappropriate precursors which can be modified with different substituentsas desired to be in the final products, and/or the final product of asynthesis according to Scheme I can be modified with differentsubstituents as desired. Placement, removal and/or inter-conversion ofdesired substituents on precursors, intermediates or penultimate productcompounds of formulas I and II can be accomplished by routine methodswell-known to those of ordinary skill in the art, as briefly overviewedin the following:

One or more hydroxyl groups, for example, can be converted to the oxoderivative by direct oxidation, which can be accomplished using anyknown method such as, for example, a Swern oxidation, or by reactionwith a metal oxidant, such as a chromium oxide (e.g., chromiumtrioxide), a manganese oxide (e.g., manganese dioxide or permanganate)or the like. Primary alcohols can be oxidized to aldehydes, for example,via Swern oxidation, or they can be oxidized to carboxylic acids (e.g.,—CO₂H), for example by reaction with a metal oxidant. Similarly, thethiols (e.g., —SR, —SH, and the like) can be converted to oxidizedsulfur derivatives (e.g., —SO₂R or the like) by reaction with anappropriate oxidant.

One or more hydroxyl groups can be converted to an ester (e.g., —CO₂R),for example, by reaction with an appropriate esterifying agent such asfor example, an anhydride (e.g., (R(CO))₂O) or an acid chloride (e.g.,R(CO)Cl), or the like. One or more hydroxyl groups can be converted to asulfonate (e.g., —SO₂R) by reaction with an appropriate sulfonatingagent such as, for example, a sulfonyl chloride (e.g., RSO₂Cl), or thelike, wherein R is any suitable substituent including, for example,organic substituents described herein. Ester derivatives also can beobtained, for example, by reacting one or more carboxylic acidsubstituents (e.g., —CO₂H) with an alkylating agent such as, forexample, a diazoalkane (e.g., diazomethane) an alkyl or aryl iodide, orthe like. One or more amides can be obtained by reaction of one or morecarboxylic acids with an amine under appropriate amide-formingconditions which include, for example, activation of a carboxylic acid(e.g., by conversion to an acid chloride or by reaction with acarbodiimide reagent) followed by coupling of the activated species witha suitable amine.

One or more hydroxyl groups can be converted to a halogen using ahalogenating agent such as, for example, an N-halosuccinamide such asN-iodosuccinamide, N-bromosuccinamide, N-chlorosuccinamide, or the like,in the presence of a suitable activating agent (e.g., a phosphine, orthe like). One or more hydroxyl groups also can be converted to ether byreacting one or more hydroxyls, for example, with an alkylating agent inthe presence of a suitable base. Suitable alkylating agents can include,for example, an alkyl or aryl sulfonate, an alkyl or aryl halide, or thelike. One or more suitably activated hydroxyls, for example a sulfonateester, and/or one or more suitably activated halides, can be convertedto the corresponding cyano, halo, or amino derivative by displacementwith a nucleophile which can include, for example, a thiol, a cyano, ahalide ion, or an amine (e.g., H₂NR, wherein R is a desiredsubstituent), or the like.

Amines can be obtained by a variety of methods known in the art, forexample, by hydrolysis of one or more amide groups. Amines also can beobtained by reacting one or more suitable oxo groups (e.g., an aldehydeor ketone) with one or more suitable amines under the appropriateconditions, for example, reductive amination conditions, or the like.One or more amines, in turn, can be converted to a number of otheruseful derivatives such as, for example, amides, sulfonamides, and thelike.

Certain chemical modifications of a compound of formula I or II can beintroduced as desired to obtain useful new variants with new or modifiedbiological properties such as: new or improved potency and/orselectivity for inhibiting Ras-mediated biological processes, improvedefficacy against a disease process such as, but not limited to, tumorcell growth, proliferation, survival, invasion and metastasis, as wellas resistance to chemotherapy, other molecularly targeted therapeutics,and radiation, as well as enhanced oral bioavailability, less toxicityin a particular host mammal, more advantageous pharmacokinetics and/ortissue distribution in a given host mammal, and the like. Therefore, thepresent invention employs methods for obtaining useful new compounds offormula I and II by applying one or more well-known chemical reactionsto a given compound to obtain a derivative wherein, for example, one ormore phenolic hydroxyl group(s) may instead be replaced by an ester,sulfonate ester or ether group; one or more methyl ether group(s) mayinstead be replaced by a phenolic hydroxyl group; one or more phenolichydroxyl group(s) may instead be replaced by an aromatic hydrocarbonsubstituent; a secondary amine site may instead be replaced by an amide,sulfonamide, tertiary amine, or alkyl quaternary ammonium salt; atertiary amine site may instead be replaced by a secondary amine; andone or more aromatic hydrogen substituent(s) may instead be replaced bya halogen, nitro, amino, hydroxyl, thiol or cyano substituent.

Depending upon the stoichiometric amount of the particular reactant, acompound of formula I or II can be substituted at one, some, or all ofthe respective available positions. For example, when such a compound isreacted with a certain amount of CH₃COCl, an acetate substituent can beintroduced a one, some, or all of the available positions, which mayinclude, for example ether or amino positions.

Other examples may include, but are not limited to: (1) conversion toester, sulfonate ester, and ether substituents at one or more phenolichydroxyl positions in compounds of formula I and II; for instance, forpreparation of esters or sulfonate esters a given compound can bereacted with an acid halide (e.g., RCOX or RSO₂X, where X is Cl, Br orI, and R is a C₁-C₆ aliphatic or aromatic radical) in anhydrous pyridineor triethylamine; alternatively, the given compound may be reacted withan acid (RCO₂H or RSO₃H) wherein R is an aliphatic or aromatic radicaland dicyclohexylcarbodiimide in triethylamine to prepare the ester orsulfonate ester; for preparation of ethers, the given compound isreacted with an organic halide (e.g., RX or RCH₂X, where X is Cl, Br orI, and R is a C₁-C₆ aliphatic or aromatic radical) in anhydrous acetonewith anhydrous potassium carbonate; (2) removal of an ether methylgroup(s) to provide a phenolic hydroxyl functionality and/or conversionof that moiety to an ester, sulfonate, or other ether in a compound orderivative of formula I or II: for instance, for hydrolytic cleavage ofa methyl ether substituent and conversion to a phenolic hydroxyl moiety,the given compound is reacted with BBr₃ or BX₃.(CH₃)₂S in CH₂Cl₂ (whereX is F, Cl or Br); the resulting phenol can be converted to an ester,sulfonate ester or ether as described above; (3) preparation of amide orsulfonamide derivatives at an amine site in a compound of formula I orII: for instance, for preparation of amides or sulfonamide derivatives,the same general procedures described above in (1) apply; in either case(procedure (1) or (3)), an appropriate functional group protectionstrategy (blocking/deblocking of selected group(s)) may need to beapplied; (4) conversion of a secondary amine functionality in a compoundof formula I or II to a tertiary amine: for instance, for preparation ofa tertiary amine, the given compound is reacted with an aldehyde, andthe resulting product is then reduced with NaBH₄; alternatively, forpreparation of an alkyl ammonium salt, the given compound is reactedwith an alkyl halide (RX, where X is Cl, Br or I, and R is a C₁-C₆aliphatic radical) in an anhydrous aprotic solvent; (5) conversion of atertiary amine functionality in a compound of formula I or II to asecondary amine; for instance, for preparation of a secondary amine, thegiven compound is reacted with cyanogen bromide to give a cyanamidederivative which is then treated with LiA1H₄; (6) conversion of one ormore phenolic hydroxyl groups in a given compound of formula I or II toan aromatic hydrogen substituent: for instance, the given compound isconverted (after suitable protection of any amine substituent(s) ifnecessary) to the triflate ester to give the corresponding deoxycompound; (7) substitution of one or more hydrogen substituent(s) on thearyl system(s) on a compound of formula I or II by halogen, nitro,amino, hydroxyl, thiol, or cyano groups: for instance, for preparationof a bromine-substituted derivative, the given compound is reacted withBr₂ in H₂O; for the preparation of other substituted derivatives, thegiven compound is treated with HNO₃/HOAc to provide a nitro-substituted(—NO₂) derivative; in turn, the nitro-derivative can be reduced to anamino derivative, and the amino derivative is the point of origin of thechloro, iodo, cyano, thiol and hydroxyl substitution via well-known andpracticed diazonium substitution reactions. More detailed, specificillustrations of synthesis and derivatization procedures that can beemployed to access any desired member of the family of compoundsrepresented by formulae I and II and derivatives thereof, are providedin the examples that follow herein.

It will be appreciated that certain compounds of formula I or II canhave one or more asymmetric carbon(s) and thus such compounds arecapable of existing as enantiomers or diastereomers. Unless otherwisespecified, the present invention includes such enantiomers ordiastereomers, including any racemates thereof. If desired, the separateenantiomers or diastereomers can be synthesized from appropriate chiralstarting materials, or the racemates can be resolved by conventionalprocedures, which are well-known to those skilled in the art, such aschiral chromatography, fractional crystallization of diastereomers ordiastereomeric salts, and the like. Certain compounds can exist asgeometrical isomers, such as, for example, compounds with double-bondedsubstituents with geometrical isomers Z and E, and the present inventionincludes all such isomers, including certain isomers, for example the Zisomers, which are preferred. Also, certain compounds may containsubstituents wherein there is restricted rotation and/or other geometricisomers are possible. For example, certain oxime substituents may existin syn or anti configurations. The present invention includes all suchconfigurations, including all possible hindered-rotational isomers, andother geometric isomers.

It will be appreciated by one skilled in the art that the proof orconfirmation of the chemical structure of a compound provided by or usedin the present invention can be demonstrated using at least one or morewell-known and established, convergent methods including, but notlimited to, for example: proton and/or carbon NMR spectroscopy, massspectrometry, x-ray crystallography, chemical degradation, and the like.

One or more compound(s) of formula I or II or pharmaceuticallyacceptable salt(s) or prodrugs(s) thereof can be included in acomposition, e.g., a pharmaceutical composition. In that respect, thepresent invention further provides a composition that includes aneffective amount of at least one compound of formula I or II, which maybe in the form of pharmaceutically acceptable salt(s) or prodrug(s)thereof and a pharmaceutically acceptable carrier. The composition ofthe present invention preferably includes a therapeutically orprophylactically effective amount of at least one Ras-inhibitorycompound of formula I or II. The therapeutically or prophylacticallyeffective amount can include an amount that produces a therapeutic orprophylactic response in a patient to whom a compound or composition ofthe present invention is administered. A therapeutically orprophylactically effective amount can include, for example, aRas-inhibitory and/or an anticancer effective amount.

The composition of the present invention can further include atherapeutically or prophylactically effective amount of at least oneadditional compound other than a compound of formula I or II, which mayor may not be another Ras-inhibitory compound, and may be an anticancercompound. When the additional compound is a Ras-inhibitory compoundother than a compound of formula I or II, it is preferably present inthe composition in a Ras-inhibiting amount. When the additional compoundis an anticancer compound in general, it is preferably present in thecomposition in an anticancer effective amount.

The composition of the present invention can be produced by combiningone or more compound(s) of formula I or II with an appropriatepharmaceutically acceptable carrier, and can be formulated into asuitable preparation, which may include, for example, preparations insolid, semi-solid, liquid or gaseous forms such as tablets, capsules,powers, granules, ointments, solutions, suppositories, injections,inhalants, and aerosols, and other formulations known in the art fortheir respective routes of administration. In pharmaceutical dosageforms, a compound of formula I or II can be used alone or in appropriateassociation, as well as in combination, with other pharmacologicallyactive compounds, including other compounds, e.g., other Ras-inhibitorycompounds, as described herein.

Any suitable pharmacologically or physiologically acceptable carrier canbe utilized. The following methods and carriers are merely exemplary andare in no way limiting. In the case of oral preparations, a compound offormula I or II can be administered alone or in combination with atherapeutically or prophylactically effective amount of at least oneother compound. The active ingredient(s) can be combined, if desired,with appropriate additives to make tablets, powders, granules, capsulesor the like.

Suitable additives can include, for example, lactose, mannitol, cornstarch or potato starch. Suitable additives also can include binders,for example crystalline cellulose, cellulose derivatives, acacia, orgelatins; disintegrants, for example, corn starch, potato starch orsodium carboxymethylcellulose; or lubricants such as talc or magnesiumstearate. If desired, other additives such as, for example, diluents,buffering agents, moistening agents, preservatives, and/or flavoringagents, and the like, can be included in the composition.

The Ras-inhibitory compounds used in accordance with the presentinvention can be formulated into a preparation for injection or infusionby dissolution, suspension, or emulsification in an aqueous ornon-aqueous solvent, such as vegetable oil, synthetic aliphatic acidglycerides, esters of higher aliphatic acid or propylene glycol (ifdesired, with conventional additives such as solubilizers isotonicagents, suspending agents, emulsifying agents, stabilizers, andpreservatives).

The compounds of formulas I and II also can be made into an aerosolformulation to be administered by inhalation. Such aerosol formulationscan be placed into pressurized acceptable propellants such asdichlorodifluoromethane, propane, nitrogen, and the like.

The compounds can be formulated into suppositories by admixture with avariety of bases such as emulsifying bases or water-soluble bases. Thesuppository formulations can be administered rectally, and can includevehicles such as cocoa butter, carbowaxes, and polyethylene glycols,which melt at body temperature but are solid at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions can be provided wherein each dosage unit, e.g.,teaspoonful, tablet, or suppository contains a predetermined amount ofthe composition containing the compound of formula I or II. Similarly,unit dosage forms for injection or intravenous administration cancomprise a composition as a solution in sterile water, normal saline, orother pharmaceutically acceptable carrier.

The term “unit dosage form” as used herein refers to physically discreteunits suitable as unitary dosages for human and animal subjects, eachunit containing a predetermined quantity of at least one compound(s) offormula I or II (alone, or if desired, with another therapeutic orprophylactic agent). The unit dosage can be determined by methods knownto those of skill in the art, for example, by calculating the amount ofactive ingredient sufficient to produce the desired effect inassociation with a pharmaceutically acceptable carrier. Thespecifications for the unit dosage forms that can be used in accordancewith the present invention depend on the particular effect to beachieved and the particular pharmacodynamics associated with thecompound(s) in the individual host.

Pharmaceutically acceptable carriers, for example, vehicles, adjuvants,excipients, or diluents, are accessible to those of skill in the art andare typically available commercially. One skilled in the art can easilydetermine the appropriate method of administration for the exactformulation of the composition being used. Any necessary adjustments indose can readily be made by an ordinarily skilled practitioner toaddress the nature or severity of the condition being treated.Adjustments in dose also can be made on the basis of other factors suchas, for example, the individual patient's overall physical health, sexage, prior medical history, and the like.

The compounds of formulas I and II can be utilized in a variety oftherapeutic and prophylactic (disease preventing) applications, and alsoin certain non-therapeutic or non-prophylactic applications. It will beappreciated that one or more of these compounds can be used, forexample, as a control in diagnostic kits, bioassays, or the like.Preferably the method of the present invention is appliedtherapeutically or prophylactically, for example, toward treatment orprevention of cancer or toward treatment or prevention of a condition(e.g. an abnormal condition or disease) treatable by the inhibition ofRas-mediated biological process(es). The compounds of formulas I and IIcan be administered alone, or in combination with a therapeutically orprophylactically effective amount of at least one additional compoundother than a compound of formula I or II.

Accordingly, the present invention further provides a method oftherapeutically or prophylactically treating a condition treatable bythe inhibition of one or more Ras-mediated biological processes, whichmethod includes administering to a patient a Ras-inhibiting amount of atleast one Ras-inhibitory compound of formula I or II. More particularly,the present invention provides a method of therapeutically orprophylactically treating a condition treatable by the inhibition of oneor more Ras-mediated biological processes, which includes administeringa Ras-inhibiting effective amount of at least one compound of formula Ior II.

A number of conditions can be treated in accordance with the method ofthe present invention. The compounds of formulas I and II and theircompositions can be used medically to regulate biological phenomena,including but not limited to such Ras-modulated processes as tumor cellgrowth, proliferation, survival, invasion and metastasis, as well asresistance to chemotherapy, other molecularly targeted therapeutics, andradiation. The compounds of formula I and II are therefore useful in thetreatment of diseases and conditions that can be controlled by theinhibition of Ras-mediated cellular functions. Such diseases include,for example, diseases wherein hyperactive Ras (e.g., including mutantRas) is implicated; such diseases prominently include cancer, amongothers. Compounds of formula I or II can be expected to have efficaciousactions in patients with cancer, especially in patients whose cancershave underlying hyperactive, over-expressed or mutant Ras-mediatedpathological processes that are inhibited by a compound(s) of formula Ior II. Other aberrant Ras-mediated diseases or conditions that areexpected to be treatable or preventable by administration ofRas-inhibiting amounts of compound(s) of formula I or II include forexample, neurofibromatosis and Costello syndrome. In the instance ofcancer particularly, compound(s) of formula I or II may promote broadersensitivity of cancer to other drugs and/or radiation therapy byinhibiting the ability of cancer cells to develop or express resistanceto such drugs and/or radiation therapy making possible the effectivechemotherapeutic and/or radiotherapeutic treatment of cancer.

In accordance with an embodiment of the method of the presentintervention, it is preferred that a Ras-inhibiting effective amount isused. In that regard, it is preferred that the Ras-inhibiting amount iseffective to inhibit one or more conditions selected from the groupconsisting of tumor cell growth, proliferation, survival, invasion andmetastasis, as well as resistance to chemotherapy, other molecularlytargeted therapeutics, and radiation.

The method of the present invention further includes administering aRas-inhibiting effective amount of at least one additional compoundother than a compound of formula I or II. In some instances, the methodof the present invention can be made more effective by administering oneor more other Ras-inhibitory compound(s), along with a compound offormula I or II. One or more Ras-inhibitory compound(s) of formula I, IIor III also can be co-administered in combination with an anticanceragent other than a compound of formula I or II, for example, to causeanticancer chemotherapy-resistant and/or radiation-resistant tumor cellsto become chemotherapy-sensitive and/or radiation-sensitive and/or toinhibit de novo the development of cancer cell resistance to theanticancer agent and/or to cancer cell resistance to radiationtreatment.

In accordance with an embodiment of the method, the patient ispre-selected by utilizing an assay of said patient's tissue, blood ortumor for an abnormal, mutant or hyperactive ras gene or Ras protein, oran aberrant Ras-mediated biological process.

In accordance with the methods of the present invention, one or morecompounds of formula I or II can be administered by any suitable routeincluding, for example, oral or parenteral, including intravenous,subcutaneous, intraarterial, intraperitoneal, ophthalmic, intramuscular,buccal, rectal, vaginal, intraorbital, intracerebral, intracranial,intraspinal, intraventricuclar, intrathecal, intracisternal,intracapsular, intrapulmonary, intranasal, transmucosal, transdermal, orvia inhalation. For example, one or more compound(s) of formula I or IIcan be administered as a solution that is suitable for intravenousinjection or infusion, or can be administered as a tablet, a capsule, orthe like, in any other suitable composition or formulation as describedherein. Accordingly, there is a wide variety of suitable formulations ofthe pharmaceutical composition of the present invention. Theformulations may also be applied topically.

The Ras-“inhibiting-effective amount” as utilized in accordance with anembodiment of the composition and method of the present invention,includes the dose necessary to achieve a Ras-“inhibiting effectivelevel” of the active compound in an individual patient. TheRas-inhibiting-effective amount can be defined, for example, as thatamount required to be administered to an individual patient to achievein said patient a Ras-inhibiting-effective blood or tissue level, and/orintracellular target-inhibiting level of a compound of formula I or IIto cause the desired medical treatment.

By way of example and not intending to limit the invention, the dose ofthe pharmaceutically active agent(s) described herein for methods ofpreventing or treating a disease or disorder can be, in embodiments,about 0.001 to about 1 mg/kg body weight of the subject being treatedper day, for example, about 0.001 mg, 0.002 mg, 0.005 mg, 0.010 mg,0.015 mg, 0.020 mg, 0.025 mg, 0.050 mg, 0.075 mg, 0.1 mg, 0.15 mg, 0.2mg, 0.25 mg, 0.5 mg, 0.75 mg, or 1 mg/kg body weight per day. In certainembodiments, the dose of the pharmaceutically active agent(s) describedherein can be about 1 to about 1000 mg/kg body weight of the subjectbeing treated per day, for example, about 1 mg, 2 mg, 5 mg, 10 mg, 15mg, 0.020 mg, 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 250 mg, 500mg, 750 mg, or 1000 mg/kg body weight per day.

The terms “treat,” “prevent,” “ameliorate,” and “inhibit,” as well aswords stemming therefrom, as used herein, do not necessarily imply 100%or complete treatment, prevention, amelioration, or inhibition. Rather,there are varying degrees of treatment, prevention, amelioration, andinhibition of which one of ordinary skill in the art recognizes ashaving a potential benefit or therapeutic effect. In this respect, theinventive methods can provide any amount of any level of treatment,prevention, amelioration, or inhibition of the disorder in a mammal. Forexample, a disorder, including symptoms or conditions thereof, may bereduced by, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%,or 10%. Furthermore, the treatment, prevention, amelioration, orinhibition provided by the inventive method can include treatment,prevention, amelioration, or inhibition of one or more conditions orsymptoms of the disorder, e.g., cancer. Also, for purposes herein,“treatment,” “prevention,” “amelioration,” or “inhibition” can encompassdelaying the onset of the disorder, or a symptom or condition thereof

When the effective level is used as the preferred endpoint for dosing,the actual dose and schedule can vary depending, for example, uponinter-individual differences in pharmacokinetics, drug distribution,metabolism, and the like. The effective level also can vary when one ormore compound(s) of formula I or II are used in combination with othertherapeutic agents, for example, one or more additional anticancercompound(s), or a combination thereof. Moreover, the effective level canvary depending upon the particular disease (e.g., cancer orneurofibromatosis) or biological process (e.g., tumor cell growth,proliferation, survival, invasion and metastasis, as well as resistanceto chemotherapy, other molecularly targeted therapeutics, and radiation)for which treatment is desired. Similarly, the effective level can varydepending on whether the treatment is for therapy or prevention of aparticular disease such as, for example, cancer.

Compounds of formula I and II can be expected to be broadly efficaciousanticancer agents, which will inhibit or destroy human solid tumors, andas well non-solid cancer such as leukemias and certain lymphomas. Solidtumors may include particularly those tumors where ras gene mutationsare highly prevalent, such as pancreatic cancer, lung cancer and coloncancer, as well as diverse other solid tumors such as, for example,melanoma, ovarian cancer, renal cancer, prostate cancer, head and neckcancer, endocrine tumors, uterine cancer, breast cancer, sarcomas,gastric cancer, hepatic cancer, esophageal cancer, central nervoussystem (e.g., brain) cancer, hepatic cancer, germline cancer, and thelike.

In a preferred embodiment of the present invention, patients who aremost likely to have a favorable response to a Ras-inhibitory compound offormula I or II can be pre-selected, prior to said treatment with saidcompound, by assaying said patient's blood, tissues or tumor for thepresence ras gene mutations and/or abnormal Ras proteins and/or aberrantRas-mediated biological function(s), using assay procedures (includinguse of commercially available assay kits) well-known to those ofordinary skill in the art.

Accordingly, the present invention further provides a method oftherapeutically or prophylactically treating cancer, which methodcomprises administering to a patient in need thereof an anticancereffective amount of at least one Ras-inhibitory compound(s) of formula Ior II. The anticancer effective amount can be determined, for example,by determining an amount to be administered effective to produce aRas-inhibiting-effective blood or tissue level and/or intracellulartarget-inhibiting “effective level” in the subject patient. Theeffective level can be chosen, for example, as that blood and/or tissuelevel (e.g., 10¹²-10⁻⁶ M from examples that follow) effective to inhibitthe proliferation of tumor cells in a screening assay. Similarly, theeffective level can be determined, for example, on the basis of theblood, tissue or tumor level in a patient that corresponds to aconcentration of a therapeutic agent that effectively inhibits thegrowth of a human cancer in any assay that is clinically predictive ofanticancer activity. Further, the effective level can be determined, forexample based on a concentration at which certain markers of cancer in apatient's blood or tumor tissue (e.g., mutant or hyperactive ras gene(s)and/or Ras protein(s) and/or aberrant Ras-mediated biologicalpathway(s)) are inhibited by a particular compound that inhibits cancer.Alternatively, the effective level can be determined, for example, basedon a concentration effective to slow or stop the growth of a patient'scancer, or cause a patient's cancer to regress or disappear, or render apatient asymptomatic to a particular cancer, or improve a cancerpatient's subjective sense of condition. The anticancer effective levelcan then be used to approximate (e.g., by extrapolation) or even todetermine precisely, the level which is required clinically to achieve aRas-inhibiting-effective blood, tissue, tumor and/or intracellular levelto cause the desired medical treatment. It will be appreciated that thedetermination of the therapeutically effective amount clinicallyrequired to effectively inhibit Ras-mediated processes also requiresconsideration of other variables that can influence the effective level,as discussed herein. When a fixed effective amount is used as apreferred endpoint for dosing, the actual dose and dosing schedule fordrug administration can vary for each patient depending upon factorsthat include, for example, inter-individual differences inpharmacokinetics, drug absorption, drug disposition and tissuedistribution, drug metabolism, drug excretion, whether other drugs areused in combination, or other factors described herein that influencethe effective level.

One skilled in the art and knowing and understanding the disclosures ofthe present invention can readily determine the appropriate dose,schedule, or method of administering a particular formulation, in orderto achieve the desired effective level in an individual patient. Giventhe disclosures herein, one skilled in the art also can readilydetermine and use an appropriate indicator of the effective level of thecompound(s) of formula I and II. For example, the effective level can bedetermined by direct analysis (e.g., analytical chemistry) or byindirect analysis (e.g., with clinical chemistry indicators) ofappropriate patient samples (e.g., blood and/or tissues). The effectivelevel also can be determined, for example, if the compound in questionhas antitumor activity, by direct or indirect observations, such as, forexample, observing the shrinkage, slowing or cessation of growth orspreading of a tumor in a cancer patient. There are many references tothe art that describe the protocols used in administering and monitoringresponses to active compounds in a patient in need thereof. For example,drug-appropriate protocols used in the administration of different typesof anticancer agents to patients are described in “Cancer Chemotherapyand Biotherapy: Principles and Practice” eds. Chabner and Longo,Lippincott, Williams and Wilkins (2011), and citations therein.

The present inventive method of therapeutically or prophylacticallytreating cancer further includes administering an anticancer effectiveamount of at least one additional compound other than a compound offormula I or II. For example, one or more compound(s) of formula I or IIcan be co-administered with an anticancer agent, and/or can beco-administered with radiation therapy, in which case the effectivelevel is the level needed to inhibit or reverse the ability of thecancer to develop resistance to the anticancer agent and/or to theradiation therapy, respectively.

Examples of anticancer compounds include reversible DNA binders, DNAalkylators, and DNA strand breakers. Examples of suitable reversible DNAbinders include topetecan hydrochloride, irinotecan (CPT11-Camptosar),rubitecan, exatecan, nalidixic acid, TAS-103, etoposide, acridines(e.g., amsacrine, aminocrine), actinomycins (e.g., actinomycin D),anthracyclines (e.g., doxorubicin, daunorubicin), benzophenainse, XR11576/MLN 576, benzopyridoindoles, Mitoxantrone, AQ4, Etopside,Teniposide, (epipodophyllotoxins), and bisintercalating agents such astriostin A and echinomycin.

Examples of suitable DNA alkylators include sulfur mustard, the nitrogenmustards (e.g., mechlorethamine), chlorambucil, melphalan,ethyleneimines (e.g., triethylenemelamine, carboquone, diaziquone),methyl methanesulfonate, busulfan, CC-1065, duocarmycins (e.g.,duocarmycin A, duocarmycin SA), metabolically activated alkylatingagents such as nitrosoureas (e.g., carmustine, lomustine,(2-chloroethyl)nitrosoureas), triazine antitumor drugs such astriazenoimidazole (e.g., dacarbazine), mitomycin C, leinamycin, and thelike.

Examples of suitable DNA strand breakers include doxorubicin anddaunorubicin (which are also reversible DNA binders), otheranthracyclines, belomycins, tirapazamine, enediyne antitumor antibioticssuch as neocarzinostatin, esperamicins, calicheamicins, dynemicin A,hedarcidin, C-1027, N1999A2, esperamicins, zinostatin, and the like.

Examples of anticancer agents include abarelix, aldesleukin,alemtuzumab, altretamine, amifostine, aminoglutethimide, anastrazole,arsenic trioxide, asparaginase, azacitidine, azathioprine, BCG vaccine,bevacizumab, bexarotene, bicalutamide, bleomycin sulfate, bortezomib,bromocriptine, busulfan, capecitabine, carboplatin, carmustine,cetuximab, chlorambucil, chloroquine phosphate, cladribine,cyclophosphamide, cyclosporine, cytarabine, dacarbazine, dactinomycin,daunorubicin hydrochloride, daunorubicin citrate liposomal, dexrazoxane,docetaxel, doxorubicin hydrochloride, doxorubicin hydrochlorideliposomal, epirubicin hydrochloride, estramustine phosphate sodium,etoposide, estretinate, exemestane, floxuridine, fludarabine phosphate,fluorouracil, fluoxymesterone, flutamide, fulvestrant, gefitinib,gemcitabine hydrochloride, gemtuzumab ozogamicin, goserelin acetate,hydroxyurea, idarubicin hydrochloride, ifosfamide, imtinib mesylate,interferon alfa-2a, interferon alfa-2b, irinotecan hydrochloridetrihydrate, letrozole, leucovorin calcium, leuprolide acetate,levamisole hydrochloride, lomustine, lymphocyte immune anti-thymocyteglobulin (equine), mechlorethamine hydrochloride, medoxyprogestoneacetate, melphalan, mercaptopurine, mesna, methotrexate, mitomycin,mitotane, mitoxantrone hydrochloride, nilutamide, oxaliplatin,paclitaxel, pegaspargase, pentostatin, plicamycin, porfimer sodium,procarbazine hydrochloride, streptozocin, tamoxifen citrate,temozolomide, teniposide, testolactone, testosterone propionate,thioguaine, thiotepa, topotecan hydrochloride, tretinoin, uracilmustard, valrubicin, vinblastine sulfate, vincristine sulfate, andvinorelbine.

Suitable forms of radiation therapy include, for example, all forms ofradiation therapy approved for commercial use in the United States, andthose forms that will become approved in the future, for which radiationresistance thereto can be controlled by a Ras-inhibitory compound offormula I or II.

In accordance with an embodiment of the methods of the presentinvention, prophylaxis includes inhibition as described herein, e.g.,inhibition of the growth or proliferation of cancer cells, or inhibitionof aberrant Ras-mediated cellular functions. The inhibition can be, butneed not be, 100% inhibition in order to be prophylactically effective,and a clinically desirable benefit can be realized with less than 100%inhibition.

The particular Ras-inhibitory compound(s) of formula I or II used inaccordance with the present invention can be selected based upon thepotency and/or selectivity for inhibiting Ras-mediated cellularprocesses, as assessed by in vitro or in vivo assays, and/or based onother pharmacological, toxicological, pharmaceutical or other pertinentconsiderations that are well-known to those skilled in the art. Routinemethods for the specific bioassay, quantitation and comparisons ofRas-inhibitory inhibitory and other biological activities and propertiesof compounds of formula I and II in various tissues, cells, organellesand other preparations, as well as in vivo testing in animals arewell-documented in the literature (e.g., see Teicher and Andrews (eds.),Anticancer Drug Development Guide, Humana (2004), and various authorsand chapters therein). More specific illustrations of these and otherdetails pertinent to enablement of the present invention are provided inthe examples which follow.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

Example 1

This example illustrates the synthesis of a compound employed in anembodiment of the invention:(Z)-2-(5-fluoro-1-(4-hydroxy-3,5-dimethoxybenzylidene)-2-methyl-1H-inden-3-yl)-N-(furan-2-ylmethyl)acetamide(007).

(A) p-fluoro-α-methylcinnamic acid: p-Fluorobenzaldehyde (200 g, 1.61mol), propionic anhydride (315 g, 2.42 mol) and sodium propionate (155g, 1.61 mol) in a 1 L three-necked flask in an atmosphere of argon wasstirred in an oil bath to 140° C. for 36 hours. The clear solution wascooled to 100° C. and poured into 8 L of water. The precipitate wascollected and dissolved by adding potassium hydroxide to 2 L of icewater to pH 12. The aqueous solution was extracted with ether, and theextracts washed with potassium hydroxide solution (200 mL×2). Thecombined aqueous solution was acidified with concentrated HCl. Theprecipitate was collected by filtration, washed with water, ethanol andhexane, dried under air to give p-fluoro-α-methylcinnamic acid, whichwas used for next step reaction without further purification.

(B) p-Fluoro-α-methylhydrocinnamic acid: A 2 L catalytic hydrogenationflask containing p-fluoro-α-methylcinnamic acid (180 g, 0.987 mol),5%-Pd/C (1.2 g) and 1.2 L ethanol was flushed with argon and warmed to65-70° C. The mixture was treated with hydrogen (40 psi) until thehydrogen uptake ceases (about 30 min). The catalyst was filtered off,and the filtrate was concentrated in vacuum to givep-fluoro-α-methylhydrocinnamic acid as an oil.

(C) 5-fluoro-2-methylindanone: Polyphosphoric acid (PPA 85%, 650 g) waswarmed in a 80° C. water bath for 1 h, then transferred to a 1 L3-necked flask equipped with a mechanical stirrer, a dropping funnel,and a thermometer. The flask was warmed in a 70° C. oil bath andp-fluoro-α-methylhydrocinnamic acid (93.2 g, 0 5 mol) was added in about5 minutes with stirring. The temperature was gradually raised to 90° C.,and kept there for about 30 min. The reaction mixture was poured into 2L of ice water, the aqueous layer extracted with ether, and the solutionwashed twice with saturated sodium chloride solution, 5% Na₂CO₃solution, water, dried over Na₂SO₄, and then concentrated to give amilky oil. The oil was dissolved in 100 mL of methylene chloride and 200mL of hexane, and the solution was loaded to a dry-packed silica gelflash column (800 g of TLC grade silica gel tightly packed in a 2 Lfritted funnel, vacuum), eluted with 5% ether-hexane to give5-fluoro-2-methylindanone as a clear oil.

(D) 5-fluoro-2-methylindenyl-3-acetic acid: A mixture of5-fluoro-2-methylindanone (184 g, 1.12 mol), cyanoacetic acid (105 g,1.23 mol), acetic acid (130 g), and ammonium acetate (34 g) in drytoluene (about 600 ml) was refluxed for 48 to 72 hours, and theliberated water/acetic acid was collected in a Dean Stark trap. To thecooled reaction mixture was added 600 mL of methylene chloride, thesolution washed with water (200 mL×3), the organic layer concentrated,and the residue treated with 150 g of potassium hydroxide in 300 ml ofethanol and 200 ml of water. The mixture was refluxed overnight undernitrogen, the ethanol removed under vacuum, 500 ml water added, theaqueous solution washed well with ether and then boiled with charcoal.The aqueous filtrate was acidified to pH 2 with 50% hydrochloric acid,and extracted with methylene chloride (300 mL×3). The solvent wasevaporated, and the residue treated with acetone in a sonicator bathuntil precipitate formed. The mixture was stored in a −20° C. freezerovernight, and the precipitate collected by filtration. The proceduregave 5-fluoro-2-methylindenyl-3-acetic acid as a colorless solid (mp164-166° C.).

(E)(Z)-2-(5-fluoro-1-(4-hydroxy-3,5-dimethoxybenzylidene)-2-methyl-1H-inden-3-yl)aceticacid: The 5-fluoro-2-methyl-3-indenylacetic acid (0.54 g, 2.62 mmol),4-acetoxy-3,5-dimethoxybenzaldehyde (0.60 g, 2.67 mmol) and potassiumbutoxide (0.69 g, 7.7 mmol) in DMSO (6 ml) were stirred in a microwavesynthesizer at 75° C. under argon for 2 h. After cooling, the reactionmixture was poured into 50 ml of ice-water, and was acidified with 2Nhydrochloric acid. The mixture was extracted with methylene chloride (25mL×2), the combined organic layer washed with water (25 mL×2), andconcentrated. The residue was purified on a silica gel column threetimes to produce the titled compound (E, 117 mg) as a yellow/orangesolid.

(F)(Z)-2-(5-fluoro-1-(4-hydroxy-3,5-dimethoxybenzylidene)-2-methyl-1H-inden-3-yl)-N-(furan-2-ylmethyl)acetamide(007): The(Z)-2-(5-fluoro-1-(4-hydroxy-3,5-dimethoxybenzylidene)-2-methyl-1H-inden-3-yl)aceticacid (120 mg, 0.324 mmol), 1,1′-Carbonyldiimidazole (100 mg, 0.61 mmol)in 5 mL of anhydrous methylene chloride was stirred for 30 min at roomtemperature. Furfuryl amine (100 1.13 mmol) was added, the reactionmixture stirred for 2 h, and quenched with 1 ml of 30% potassiumhydroxide solution, which was diluted with 25 mL of methylene chloride,neutralized with 2 mL of acetic acid, washed with water (20 mL×3), driedwith sodium sulfate, then concentrated. The residue was purified withsilica gel column, eluted with hexane/acetone. The major yellow fractionwas collected and after concentration the residue (120 mg) was storedunder argon in a freezer, and after 2 weeks crystals formed. The mixturecontaining the crystals was suspended in 2 mL of ethyl ether and 3 mL ofhexane, treated with sonicator for 1 h, then stored in a −20° C. freezerovernight. The precipitate was collected by filtration, and the titledcompound (007) was obtained as a yellow solid (41 mg).

Example 2

This example illustrates the synthesis of another compound employed inan embodiment of the invention:(Z)-2-(5-methoxy-1-(4-hydroxy-3,5-dimethoxybenzylidene)-2-methyl-1H-inden-3-yl)-N-(furan-2-ylmethyl)acetamide(006).

(A) p-methoxy-α-methylcinnamic acid: p-Methoxybenzaldehyde (219 g, 1.61mol), propionic anhydride (315 g, 2.42 mol) and sodium propionate (155g, 1.61 mol) in a 1 L three-necked flask in an atmosphere of argon wasstirred in an oil bath at 140° C. for 36 hours. The clear solution wascooled to 100° C., poured into 8 L of water, and the precipitatecollected and dissolved by adding potassium hydroxide to 2 L of icewater to pH 12. The aqueous solution was extracted with ether, theextracts washed with potassium hydroxide solution (200 mL×2) and thecombined aqueous solution acidified with concentrated HCl. Theprecipitate was collected by filtration, washed with water, ethanol andhexane, then dried over air to give p-methoxy-α-methylcinnamic acid (235g) which was used for next step reaction without further purification.

(B) p-methoxy-α-methylhydrocinnamic acid: A 2 L catalytic hydrogenationflask containing p-methoxy-α-methylcinnamic acid (192 g, 1.00 mol),5%-Pd/C (1.2 g) and 1.2 L ethanol was flushed with argon and warmed to70° C. The mixture was treated with hydrogen (40 psi) until the hydrogenuptake ceased (about 30 min). The catalyst was filtered off, and thefiltrate concentrated in vacuum to give p-methoxy-α-methylhydrocinnamicacid as an oil.

(C) 5-methoxy-2-methylindanone: Polyphosphoric acid (PPA 85%, 650 g) waswarmed in a 60° C. water bath for 1 h, and transferred to a 1 L 3-neckedflask equipped with a mechanical stirrer, a dropping funnel, and athermometer. The flask was warmed to 50° C. in oil bath andp-methoxy-α-methylhydrocinnamic acid (96 g, 0.50 mol) was added in about5 minutes with stirring. The temperature was gradually raised to 70° C.for about 15 min, and the solution was poured into 2 L of ice water. Theaqueous layer was extracted with ether, and the solution was washedtwice with saturated sodium chloride solution, 5% Na₂CO₃ solution,water, dried over Na₂SO₄, and then concentrated to give a milky oil. Theoil was dissolved in 100 mL of methylene chloride and 200 mL of hexane,and applied to a dry-packed silica gel flash column (800 g of TLC gradesilica gel tightly packed in a 2 L fritted funnel, vacuum), eluted with5% ether-petroleum ether to give 6-methoxy-2-methylindanone as a clearoil.

(D) 5-methoxy-2-methylindenyl-3-acetic acid: A mixture of6-methoxy-2-methylindanone (197 g, 1.12 mol), cyanoacetic acid (105 g,1.23 mol), acetic acid (130 g), and ammonium acetate (34 g) in drytoluene (about 600 ml) was refluxed for 48 to 72 hours, until theliberated water collected in a Dean Stark trap ceased. To the cooledtoluene reaction mixture was added 600 mL of methylene chloride. Thesolution was washed with water (200 mL×3), the organic layerconcentrated, and the residue treated with 150 g of potassium hydroxidein 300 ml of ethanol and 200 ml of water. The mixture was refluxedovernight under nitrogen, the ethanol removed under vacuum, 500 ml wateradded, the aqueous solution washed well with ether and then boiled withcharcoal. The aqueous filtrate was acidified to pH 2 with 50%hydrochloric acid, extracted with methylene chloride (300 mL×3), solventevaporated, and the residue treated with acetone in a sonicator bathuntil a precipitate formed. The mixture was stored at −20° C. overnight,and the precipitate collected by filtration. The procedure gave5-methoxy-2-methylindenyl-3-acetic acid as a colorless solid (mp164-166° C.).

(E)(Z)-2-(5-methoxy-1-(4-hydroxy-3,5-dimethoxybenzylidene)-2-methyl-1H-inden-3-yl)aceticacid: 5-methoxy-2-methyl-3-indenylacetic acid (0.50 g, 2.29 mmol),4-acetoxy-3,5-dimethoxybenzaldehyde (0.60 g, 2.67 mmol) and potassiumbutoxide (1.0 g, 8.9 mmol) in anhydrous DMSO (5 ml) and pyridine (10 mL)were stirred at 95° C. under argon for 1 h. The reaction mixture wascooled to 65° C. 1.0 mL of methanol was added and stirred for 30 min.After cooling, the mixture was poured into 50 ml of ice-water, acidifiedwith 2N hydrochloric acid, and extracted with methylene chloride (25mL×2), and the combined organic layer washed with water (25 mL×2), andconcentrated. The residue was purified on a silica gel column twice, thefirst eluted with methylene chloride/methanol, followed by washing withhexane, acetone/acetic acid, to produce the title compound (E, 152 mg)as a yellow/orange solid.

(F)(Z)-2-(5-methoxy-1-(4-hydroxy-3,5-dimethoxybenzylidene)-2-methyl-1H-inden-3-yl)-N-(furan-2-ylmethyl)acetamide(006):(Z)-2-(5-methoxy-1-(4-hydroxy-3,5-dimethoxybenzylidene)-2-methyl-1H-inden-3-yl)aceticacid (123 mg, 0.324 mmol), 1,1′-carbonyldiimidazole (100 mg, 0.61 mmol)in 5 mL of anhydrous methylene chloride was stirred for 30 min at roomtemperature, furfuryl amine (100 μL, 1.13 mmol) was added, and thereaction mixture stirred for 2 h, then quenched with 1 ml of 30%potassium hydroxide solution. The solution was diluted with 25 mL ofmethylene chloride, neutralized with 2 mL of acetic acid, washed withwater (20 mL×3), dried with sodium sulfate, and concentrated. Theresidue was purified over silica gel, eluted with hexane/acetone, andthe major yellow fraction was collected. After concentrating, theresidue was stored in a freezer under argon until a crystal seed formed(˜2 weeks). The mixture was suspended in 2 mL of ethyl ether and 3 mL ofhexane, treated with sonicator for 1 h, then stored in a −20° C. freezerovernight. The precipitate was collected by filtration. The titlecompound (006) was obtained as a yellow solid (36 mg).

Example 3

This example illustrates the synthesis of yet another compound employedin an embodiment of the invention:(Z)-2-(1-(4-hydroxy-3,5-dimethoxybenzylidene)-5-methoxy-2-methyl-1H-inden-3-yl)-N-(pyridin-3-yl)acetamide(004).

(F)(Z)-2-(5-methoxy-1-(4-hydroxy-3,5-dimethoxybenzylidene)-2-methyl-1H-inden-3-yl)aceticacid (123 mg, 0.324 mmol) (see (E) under Example 2 above),1,1′-carbonyldiimidazole (100 mg, 0.61 mmol) in 5 mL of anhydrousmethylene chloride was stirred for 30 min at room temperature.3-Aminopyridine (100 mg, 1.04 mmol) in 4 mL of pyridine was added,stirred for 2 h at 50° C., and quenched with acetic anhydride (0.5 mL)to remove excess amine. After 30 min, 3 ml of 30% potassium hydroxidesolution was added and the solution diluted with 25 mL of methylenechloride, neutralized with 2 mL of acetic acid, washed with water (20mL×3), dried with sodium sulfate, then concentrated. The residue wasloaded on silica gel column and eluted with hexane/acetone. The majoryellow fraction was collected and recrystallized from methylene chlorideand hexane to give the title compound (004) as a yellow solid (89 mg).

Example 4

This example illustrates the synthesis of a further compound employed inan embodiment of the invention:(Z)-2-(5-fluoro-1-(4-hydroxy-3,5-dimethoxybenzylidene)-2-methyl-1H-inden-3-yl)-N-((1-methyl-1H-pyrrol-2-yl)methyl)acetamide(019).

(Z)-2-(5-fluoro-1-(4-hydroxy-3,5-dimethoxybenzylidene)-2-methyl-1H-inden-3-yl)aceticacid (120 mg, 0.324 mmol) (see (E) under Example 1 above), and1,1′-carbonyldiimidazole (100 mg, 0.61 mmol) in 5 mL of anhydrousmethylene chloride was stirred for 30 min at room temperature. To thereaction mixture (1-methyl-1H-pyrrol-2-yl)methanamine (110 1.0 mmol) and0.5 mL of pyridine were added. The mixture was stirred for 2 h, quenchedwith 1 ml of 30% potassium hydroxide solution, diluted with 25 mL ofmethylene chloride, neutralized with 2 mL of acetic acid, washed withwater (20 mL×3) and dried with sodium sulfate. The organic layer wasconcentrated and the residue was purified with silica gel column elutedwith hexane/acetone. The major yellow fraction was collected, and afterconcentration the residue was treated with acetone/hexane, sonicated for1 h, then stored in a −20° C. freezer for 2 h. The precipitate wascollected by filtration, and the titled compound (019) was obtained as ayellow solid (75 mg).

Example 5

This example illustrates the synthesis of various other exemplarycompounds of formula I used in embodiments of the invention. When thesame synthetic approach (e.g., Scheme I) is used as illustrated inExamples 1-3, except in the acetamide-forming step (F) using theappropriate precursor acetic acid derivative reacted with instead of theamine used in Example 3 one of the following amines to produce thecorresponding amides (designated in parentheses): furfuryl amine (001,008, 012, 013, 014, 016, 017, 020, 021); N,N-dimethylaminoethylamine(003); 3-aminomethylpyridine (009); 2-aminomethylpyridine (010);benzylamine (015); (1H-pyrrol-2-yl)methanamine (018, 022);(1-methyl-1H-pyrrol-2-yl)methanamine (019).

Example 6

This example illustrates synthesis of compound (029):(Z)-2-(5-fluoro-1-(4-mesyloxy-3,5-dimethoxybenzylidene)-2-methyl-1H-inden-3-yl)-N-(furan-2-ylmethyl)acetamide(029).

(Z)-2-(5-fluoro-1-(4-hydroxy-3,5-dimethoxybenzylidene)-2-methyl-1H-inden-3-yl)-N-(furan-2-ylmethyl)acetamide(007) (139 mg, 0.31 mmol), methanesulfonic anhydride (80 mg, 0.46 mmol)in 3 mL of anhydrous pyridine were stirred at 50° C. for 3 h. Thereaction mixture was quenched with water, diluted with 20 mL ofmethylene chloride, the organic solution washed with 10 mL water threetimes, and concentrated. The residue was purified on a silica gelcolumn, eluted with hexane/acetone. The major yellow fraction wascollected and recrystallized from dichloromethane and hexane to give thetitled compound (029) as a yellow solid (78 mg).

Example 7

This example illustrates the synthesis of an exemplary prodrug of acompound of formula I, specifically(Z)-2-(5-fluoro-1-(4-formoxy-3,5-dimethoxybenzylidene)-2-methyl-1H-inden-3-yl)-N-(furan-2-ylmethyl)acetamide(002, which is a prodrug of 007), which is employed in an embodiment ofthe invention.

In a sealed 25 mL round-bottom flask(Z)-2-(5-fluoro-1-(4-hydroxy-3,5-dimethoxybenzylidene)-2-methyl-1H-inden-3-yl)-N-(furan-2-ylmethyl)acetamide(007) (90 mg, 0.2 mmol) was dissolved in 4 mL of pyridine. Aceticanhydride (0.2 mL) was added. The solution was stirred at 50° C. for 2h. The reaction mixture was quenched with ethanol, concentrated, and theresidue purified on a silica gel column eluted with hexane/acetone. Themajor yellow fraction was collected, and recrystallized fromacetone/hexane to give the title compound (002) as a yellow solid.

Example 8

This example illustrates synthesis of compound 035:(Z)-2-(5-fluoro-1-(4-hydroxy-3,5-dimethoxybenzylidene)-2-methyl-1H-inden-3-yl)-N-phenylacetamide,which is employed in an embodiment of the invention.

(Z)-2-(5-fluoro-1-(4-hydroxy-3,5-dimethoxybenzylidene)-2-methyl-1H-inden-3-yl)aceticacid (120 mg, 0.324 mmol) and 1,1′-carbonyldiimidazole (100 mg, 0.61mmol) were stirred in 5 mL of anhydrous methylene chloride for 30 min atroom temperature. To the reaction mixture, aniline (93 mg, 1.0 mmol) and0.5 mL of pyridine were added. The mixture was stirred at 45° C. for 2h, quenched with 1 ml of 30% potassium hydroxide solution, diluted with25 mL of methylene chloride, acidified with acetic acid, washed withwater (20 mL×3) and dried with sodium sulfate, and the organic layerconcentrated. The residue was purified over silica gel eluted withhexane/acetone, the major yellow fraction collected, concentrated, andthe residue recrystallized from acetone/ethyl ether to give titledcompound (035) as a yellow solid (45 mg).

Example 9

Table 1 provides the ¹H-NMR data of exemplary compounds employed in theinvention. All spectra were recorded using DMSO-d⁶ as solvent.

TABLE 1 ¹H-NMR data of compounds in accordance with an embodiment of theinvention Cpd. No. NMR (δ ppm) 002 8.603 (t br, 1H, J = 5.62 Hz, CONH),8.521 (s, 1H, HCO), 7.550 (d, 1H, 7.57 Hz, furanH), 7.549 (s, 1H, IndH),7.383 (dd, 1H, J1 = 8.30 Hz, J2 = 5.37 Hz, indH), 7.285 (s, 1H, furanH),7.099 (dd, 1H, J1 = 9.68 Hz, J2 = 2.20 Hz, indH), 6.945 (s, 2H, PhH),6.910 (s, 1H, ═CH), 6.763 (m, 1H, furanH), 4.267 (d, 1H, J = 5.61 Hz,NCH₂), 3.767 (s, 6H, OCH₃), 3.460 (s, 2H, CH₂), 2.182 (s, 3H, CH₃) 00411.109 (br s, 1H, OH), 9.082 (d, 1H, J = 1.96 Hz, PyrH), 8.482 (d, 1H, J= 4.88 Hz, PyrH), 8.375 (d, 1H, J = 8.32 Hz, PyrH), 7.774 (dd, 1H, J₁ =4.63 Hz, J₂ = 8.43 Hz PyrH), 7.123 (s, 1H, Ind H) 7.520 (d, 1H, J = 8.30Hz, Ind H), 6.912 (d,1H, J = 1.97 Hz, ═CH), 6.846 (s, 2H, PhH), 6.690(br s,1H, NH), 6.537 (dd,1H, J₁ = 2.20 Hz, J₂ = 8.55 Hz, Ind H), 3.749(s, 6H, OCH₃), 3.716 (s, 3H, OCH₃), 3.378 (s, 2H, CH₂), 2.182 (s, 3H,CH₃) 006 8.714 (s br, 1H, OH), 8.549 (t, 1H, NH), 7.480 (d, 1H, J = 8.54Hz, FuranH), 7.071 (s, 1H, indH), 6.857 (d, 1H, J = 7.08 Hz, indH),6.833 (s, 2H, PhH), 6.48 (d, 1H, indH), 6.371 (m, 1H, furanH), 6.122 (m,1H, furanH), 6.175 (d, 1H, J = 2.93 Hz, ═CH), 4.261 (d, 2H, J = 5.37 Hz,NCH₂), 3.745 (s, 6H, CH₃O), 3.707 (s, 3H, CH₃O), 3.429 (s, 2H, CH₂),2.162 (s, 3H, CH₃) 007 8.792 (s br, 1H, OH), 8.574 (t, 1H, J = 5.62 Hz,NH), 7.559 (d, 1H, J = 4.88 Hz, furanH), 7.233 (s, 1H, indH), 7.084 (dd,1H, J1 = 9.22 Hz, J2 = 2.20 Hz, indH), 6.841 (s, 2H, PhH), 6.752 (1H, m,indH), 6.481 (m, 1H, furanH), 6.322 (m, 1H, furanH), 6.205 (d, 1H, J =2.93 Hz, ═CH), 4.264 (d, 2H, J = 6.17 Hz, NCH₂), 3.747 (s, 6H, MeO),3.448 (s, 2H, COCH₂), 2.16 (s, 3H, CH₃) 008 8,68 (s br, 1H, OH), 8.556(t, 1H, J = 5.37 Hz, NH), 7.539 (d, 1H, J = 4.88 Hz, furanH), 7.196 (s,1H, indH), 7.088 (s, 1H, indH), 6.915 (s, 1H, ═CH), 6.846 (s, 2H, PhH),6.37 (m, 1H, furanH), 6.198 (d, 1H, J = 2.93 Hz, furanH), 4.264 (d, 2H,J = 5.38 Hz, NCH₂), 3.752 (s, 6H, OCH₃), 3.713 (s, 3H, OCH₃), 3.493 (s,3H, OCH₃), 3.414 (s, 2H, COCH₂), 2.16 (s, 3H, CH₃) 009 8.732 (s br, 1H,OH), 8.615 (t br, 1H, J = 5.61 Hz, NH), 8.460 (s, 1H, PyrH), 8.428 (d,1H, J = 3.91 Hz, Pyr H), 7.625 (d, 1H, J = 7.81 Hz, indH), 7.483 (d, 1H,J = 8.30 Hz, indH), 7.315 (dd, 1H, PyrH), 7.081 (s, 1H, indH), 6.835 (s,2H, PhH), 6.505 (d, 1H, J = 7.3 Hz, ═CH), 4.294 (d, 2H, J = 5.37 Hz,NCH₂), 3.745 (s, 6H, OCH₃), 3.692 (s, 3H, OCH₃), 3.465 (s, 3H, OCH₃),3.414 (s, 2H, COCH₂), 2.16 (s, 3H, CH₃) 010 8.793 (s br, 1H, OH), 8.700(t br, 1H, J = 5.86 Hz, NH), 8.460 (s, 1H, PyrH), 8.488 (d, 1H, J = 3.91Hz, Pyr H), 7.711 (td, 1H, J1 = 7.57 Hz, J2 = 1.61 Hz, indH), 7.553 (dd,1H, J = 8.30 Hz, J2 = 8.26 Hz, PyH), 7.260 (s, 1H, indH), 7.241 (s,1H,═CH), 7.130 (dd, 1H, J1 = 9.52 Hz, J2 = 2.20 Hz, IndH), 6.841 (s, 2H,PhH), 6.758 (m, 1H, PyrH), 4.365 (d, 2H, J = 5.84 Hz, NCH₂), 3.745 (s,6H, OCH₃), 3.523 (s, 3H, CH₃), 3.465 (s, 3H, OCH₃), 3.414 (s, 2H,COCH₂), 2.16 (s, 3H, CH₃) 011 8.802 (s br, 1H, OH), 8.644 (t br, 1H, J =5.63 Hz, NH), 8.467 (s, H, PyrH), 8.440 (dd, 1H, J = 4.64 Hz, J2 = 1.22Hz, PyrH), 7.640 (d, 1H, J = 7.81 Hz, indH), 7.556 (dd, H, J1 = 8.54 Hz,J2 = 8.24 Hz, PyrH), 7.330 (dd, 1H, PyrH), 7.241 (s, 1H, indH), 7.064(dd, 1H, ═CH), 6.841 (s, 2H, PhH), 6.758 (m, 1H, PyrH), 4.296 (d, 2H, J= 5.62 Hz, NCH₂), 3.745 (s, 6H, OCH₃), 3.483 (s, 2H, CH₂), 2.146 (s, 3H,CH₃) 012 9.335 (s br, 1H, OH), 8.541 (t br, 1H, J = 5.61 Hz, CONH),7.552 (d, 1H, J = 0.97 Hz), 7.423 (d, 1H, J = 8.55 Hz, PhH), 7.106 (d,1H, J = 1.72 Hz), 7.064 (s, 1H), 6.986 (dd, 1H, J1 = 8.30 Hz, J2 = 1.47Hz,), 6.851 (s, 1H, PhH), 6.841 (d, 1H, J = 8.02 Hz), 6.480 (d, 1H, J1 =8.58 Hz, J2 = 2.44 Hz), 6.372 (dd, 1H, J1 = 2.93 Hz, J2 = 1.83 Hz),6.197 (d, 1H, J = 2.69 Hz), 4.254 (d, 2H, J = 5.62 Hz, NCH₂), 3.748 (s,3H, CH₃O), 3.703 (s, 3H, CH₃O), 3.424 (s, 2H, CH₂), 2.162 (s, 3H, CH₃)015 8.796 (s br, 1H, OH), 8.592 (t br, 1H, J = 5.86 Hz, CONH), 7.578(dd, 1H, IndH), 7.16-7.33 (m, 5H, phenyl H), 7.102 (dd, 1H, indH), 6.843(s, 1H, ═CH), 6.76 (m, 1H, IndH), 4.273 (d, 2H, J = 5.76 Hz, NCH2),3.747 (s, 6H, OCH₃), 3.483 (s, 2H, CH₂), 2.182 (s, 3H, CH₃) 018 10.575(s br, 1H, NH), 9.740 (s br, 1H, OH), 8.342 (t br, 1H, J = 5.37 Hz,CONH), 7.332 (d, 1H, J = 8.30 Hz, indH), 7.108 (s, 2H, PhH), 7.029 (s,1H, IndH), 6.874 (d, 1H, J = 2.27 Hz, ═CH)), 6.616 (m, 1H, PyrroH),6.496 (dd, 1H, J1 = 8.30 Hz, J2 = 2.20 Hz, IndH), 5.906 (dd, 1H,PyrroH), 5.871 (s, 1H, PyrroH), 4.188 (d, 1H, J = 5.12 Hz, CONH), 3.805(s, 3H, OCH₃), 3.703 (s, 3H, OCH₃), 3.418 (s, 2H, CH₂), 2.18 (s, 3H,CH₃) 022 10.582 (s br, 1H, NH), 8.520 (s br, 1H, OH), 8.342 (t br, 1H,CONH), 7.820 (s, 1H, indH), 7.276 (d, 1H, J = 8.30 Hz, IndH), 6.995 (s,2H, PhH), 6.889 (d, 1H, PyrroH), 6.620 (s, 1H, ═CH), 6.519 (d, 1H, J =8.30 Hz, IndH), 5.911 (d, 1H, PyrroH), 5.878 (d, 1H, PyrroH), 4.197 (d,1H, J = 4.87 Hz, CONH), 3.795 (s, 6H, OCH₃), 3.706 (s, 3H, OCH₃), 3.438(s, 2H, CH₂), 2.18 (s, 3H, CH₃) 028 8.717 (s br, 1H, OH), 8.645 (t br,1H, NH), 8.482 (d, 1H, J = 5.68 Hz PyrH), 7.698 (td, 1H, J = 7.57 Hz, J2= 1.72 Hz, PyrH), 7.490 (1H, d, J = IndH), 7.247 (d, 1H, J = 7.56 Hz,IndH), 7.245, (m, 1H, PyrH), 7.081 (s, 1H, IndH), 6.880 (s, m, 1H, ═CH),6.837 (s, 2H, PhH), 6.509 (dd, 1H, J₁ = 7.30 Hz, J₂ = 2.18 Hz, PyrH),4.362 (d, 2H, J = 5.86 Hz, NCH₂), 3.745 (s, 6H, OCH₃), 3.711 (s, 3H,OCH₃), 3.504 (s, 2H, CH₂), 2.146 (s, 3H, CH₃) 095 8.86 (br t, 1H, CONH),7.56 (d, 1H, furanH), 7.32 (dd, 1H, indH), 7.28 (s, 1H, ═CH), 7.10 (dd,1H, indH), 6.86 (s, 2H, PhH), 6.78 (td, 1H, indH), 6.38 (dd, 1H,furanH), 6.21 (d, 1H, furan), 4.26 (q, 2H, CH₂Me), 4.23 (d, 2H, CH₂N),3.77 (s, 6H, CH₃O), 2.15 (s, 3H, CH₃), 1.26 (s, 3H, CH₃)

Example 10

This example illustrates cell growth assays employed in the presentinvention. Cells used in such assays included A-549, HT-29, MDA-MB-231,Colo-205, Caco2, HCT-116, SW-480, and DLD-1 human cancer cells obtainedfrom the American Type Culture Collection (ATCC).

Human tumor cells were cultured using standard methods in RPMI-1640growth medium supplemented with 5% fetal bovine serum (FBS). Normal ratkidney (NRK) and Ki-Ras transformed NRK cells (K-NRK) were obtained fromATCC, and were cultured according to supplier recommendations.CellTiter-Glo ATP cell growth assay reagents were obtained from Promegaand used according to the manufacturer's protocol. Inhibitors of EGFR,Raf, and MEK were obtained from Selleck Chemicals. Cells were plated ata density of 5,000 cells per well in 96-well microplates or 1,250 cellsper well in 384-well plates, and allowed to attach for at least 4 h.Test compounds were dissolved in dimethyl sulfoxide (DMSO), and thisworking stock was further diluted in growth medium for addition to cellcultures. Serial dilutions of the test compound were prepared in growthmedium containing an equal amount of DMSO not exceeding 0.2% finalconcentration. Each compound concentration was tested in at least 3separate samples per cell line. At the end of a 3-day treatment period,growth inhibition was analyzed using a bioluminescent assay of ATPconcentration (Promega CellTiter-Glo) according to the manufacturer'sprotocol. Resulting luminescence was measured using the luminescencecartridge of the Molecular Devices Spectramax Paradigm microplatereader. Relative growth inhibition for each sample was determined bycomparison with the values obtained for vehicle treated control samples.Growth inhibition values were plotted with the GraphPad Prism5 softwareusing the 4-parameter logistic fit to obtain IC₅₀ values, whichcorresponds to the growth inhibitory potency of the compound.

Example 11

This example illustrates a Ras binding domain assay used in anembodiment of the present invention to measure Ras activation status.The activation state of Ras in cell lines was assayed using the ActiveRas Pull-Down and Detection Kit (Thermo Scientific). Cell lines werecultured as described above. Cells were disrupted with non-ionicdetergent, and the active (GTP-bound) Ras was isolated by its highaffinity for Raf via precipitate with sepharose-bound GST-Raf fusionprotein. The precipitated active Ras was then subjected topolyacrylamide gel electrophoresis (PAGE) and transferred tonitrocellulose membrane (western blot). Detection was achieved using theanti-Ras mouse primary antibody and anti-mouse-horseradish peroxidaseconjugated secondary antibody. Paired samples of whole cell lysate wereanalyzed by western blot for expression level of total Ras protein aswell as a gel loading control, glyceraldehyde 3-phosphate dehydrogenase(GAPDH). Digital enhanced chemiluminescence imaging of the resultingwestern blots was performed using a Syngene G:Box. The intensity of Rasbands from each cell line and the corresponding GAPDH bands werequantitated using NIH ImageJ, and expressed as “relative Rasactivation.”

Example 12

This example illustrates effects of exemplary compounds of the presentinvention on cancer cells having activated or mutated Ras compared towild-type (WT) Ras. Compounds were tested for their ability to inhibitthe growth of cells expressing constitutively active K-Ras oncogenecompared with cells with inactive Ras. A549 lung cancer cells, whichharbor a mutation in the K-Ras oncogene were treated for three days withthe compounds. As indicated in Table 2, the IC₅₀ values of the compoundsranged from <1 nM to over 5900 nM in cells with mutated Ras. Incontrast, when these compounds were tested for their ability to inhibitthe growth of colon cancer cells having the wild-type Ras protein(HT29), these compounds were significantly less potent, with IC₅₀ valuesranging from 280 nM to over 8000 nM. The ratio of a given compound'spotency (IC₅₀) to inhibit the growth of cells lacking activated Ras(HT-29) relative to that of cells with mutated or activated Ras (A549)demonstrates selectivity for the Ras-mutant-containing cells.

TABLE 2 Growth inhibition of cells with mutant Ras (N.D., notdetermined; W.T., wild type). A549 HT-29 (Ras-activated) (WT Ras)Selectivity Cpd # IC₅₀ (nM) IC₅₀ (nM) HT-29/A549 001 716 6,280 9 0033,650 4,120 1 004 79 916 12 005 5,940 7,190 1 006 11 8,130 739 007 0.86280 326 008 167 1,540 9 009 353 4,830 14 010 170 712 4 011 15 2,940 196012 170 900 5 013 300 730 2 014 110 500 5

Example 13

This example illustrates the levels of Ras activation in differentcolorectal cancer cells. In particular, a panel of human colorectalcancer cell lines was selected to further describe the selectivity ofthe compounds for cells with activated Ras. Three of the cell lines inthe panel have been reported to harbor ras mutations: HCT-116, DLD-1,and SW-480 (Stoneman and Morris, Clin Mol Pathol., 48, M326-332(1995)).Three of the cell lines were reported to express wild type Ras: HT-29,Caco-2, and Colo-205(Stoneman and Morris, supra; Shirasawa et al.,Science, 260, 85-88 (1993)). The activation state of Ras in the celllines was assayed using the Active Ras Pull-Down and Detection Kit. Theratio of the intensity of the Ras to GAPDH bands was expressed asrelative Ras activation, which is presented above each lane in FIG. 4.This experiment demonstrated that the level of Ras activation inHCT-116>DLD-1≈SW-480>Caco2>HT-29>Colo205.

Example 14

This example illustrates selective growth inhibition in colorectal celllines constitutively containing activated Ras or wild-type Ras. Thegrowth inhibitory activity of the compounds was tested using theCellTiter-Glo assay. Cells were seeded in 384-well plates and allowed toattach, then ten-fold serial dilutions of compounds were tested. Eachconcentration was tested in at least 3 separate samples per cell line.As indicated in Table 3, the potency of the compounds ranged from <1 nMin cells with Ras activation, to greater than 1300 nM in cells with WTRas. The ratio of a given compound's potency ((IC50 (subscript 50)) toinhibit the growth of cells having wild-type Ras (Caco-2) relative tothat of cells having activated Ras (SW-480) demonstrates selectivity forthe Ras-mutant-containing cells.

TABLE 3 Selective growth inhibition for active Ras in a panel ofcolorectal cancer cell lines. Caco-2 SW-480 (WT (Ras Ras) Activated IC₅₀Selectivity Cpd # IC₅₀ (nM) (nM) Caco-2/SW-480 001 435 1,100 3 004 8771,600 2 006 2.85 428 150 007 0.37 1.97 5 008 139 1,330 10 009 223 2,61012 010 2.1 33.8 16 011 21.3 47.6 2

Example 15

This example illustrates the correlation between the sensitivity tothese compounds and Ras activation status. The log of the IC₅₀ values offour highly active compounds were plotted on the y-axis of the graphsand the log of the relative levels of Ras activation (e.g., as describedin Example 9) was plotted on the x-axis. Linear regression analysis ofthe resulting coordinates demonstrates a statistically significantinverse correlation between Ras activation and sensitivity of the celllines as shown in FIG. 6, in which HCT-116, DLD-1, and SW-480 cellsharbor Ras mutations, while: HT-29, Caco-2, and Colo-205 expresswild-type Ras. Correlation coefficients (r²) are presented in each ofthe plots.

Example 16

This example illustrates use of well-established human colon tumor celllines with widely divergent Ras activation status to determineselectivity values for exemplary compounds of the present invention.Cell lines thus employed in this example were HCT-116, a highlyRas-driven line expressing mutant Ras, and HT-29, a non-Ras-driven lineexpressing wild-type, non-mutated Ras. Cells were plated at 5000cells/well in 96-well plates, and viable cell numbers were measuredusing the Cell Titer Glo ATP luminescence assay (Promega). FIGS. 6A-6Gshow results of these studies for exemplary compounds 006, 007, 019,029, 002, 010, 011, 015, 022 and 035, respectively. The calculatedHT-29/HCT-116 selectivity values for the aforementioned compounds were43, 92, 188, 35, 112, 141, 168, 80, 117, and 31, respectively. Theseselectivity values are the ratios of each compound's potency (IC₅₀) toinhibit the growth of cells lacking activated Ras ((HCT-29) relative tothat of cells with activated Ras (HCT-116), demonstrating selectivityfor the Ras-mutant-containing cells.

This example furthermore shows that prior art studies (e.g., U.S. Pat.Nos. 6,063,818 and 6,121,321) employing individual cell lines such asHT-29 or SW-480 could not have revealed compounds with Ras-selectiveactivity useful for Ras-directed medical treatments or preventions.Employment of a cell line having normal, non-mutant Ras (e.g., HT-29)concurrently with at least one or more cell line(s) having hyperactiveor mutant Ras (e.g., HCT-116 and/or SW-480) in comparative assays oftumor cell growth inhibition are required to demonstrateRas-selectivity, and to enable selection of a Ras-inhibitory compoundnecessary for the novel methods disclosed in the present invention.

Example 17

This example illustrates non-selective growth inhibition with known Raspathway inhibitors which are not compounds of the present invention. Inparticular, the growth inhibitory activity of commercially availablecompounds which are active in the Ras signal transduction pathway weretested in the same panel of cell lines using the CellTiter-Glo assay.Cells were seeded in 384-well plates and allowed to attach. Ten-foldserial dilutions of compounds were tested. Each compound concentrationwas tested in at least 3 separate samples per cell line. As indicated inTable 4 below, the potency of the EGF receptor inhibitor compoundsranged from 4 μM to >20 with no pattern of selectivity with regard toRas activation. Likewise, the C-Raf inhibitor, GW5074, did not showselectivity for cell lines expressing activated Ras. The B-Rafinhibitors tested were generally active in the low micromolar range, butwere significantly more potent in Colo-205 cells, which have the lowestlevel of active Ras. The MEK inhibitor, Selumetinib was also most potentagainst COLO-205 and HT-29 cell lines showing, if anything, a “reverse”selectivity toward inactive Ras compared with the compounds of thisinvention.

TABLE 4 Non Ras-selective growth inhibition with known Ras pathwayinhibitors. HCT-116 DLD-1 SW-480 Caco-2 HT-29 COLO-205 IC₅₀ IC₅₀ IC₅₀IC₅₀ IC₅₀ IC₅₀ Compound Target (nM) (nM) (nM) (nM) (nM) (nM) GefitinibEGFR 12,600 7,550 8,630 8,920 6,370 8,200 GW5074 C-Raf 15,800 7,7508,070 >20,000 19,100 7,100 GDC0879 B-Raf 8,140 7,640 >20,000 >20,00023,600 75.8 Vemurafenib B-Raf 5,990 5,620 6,860 5,200 5,100 151Selumetinib MEK 4,110 20,400 >20,000 >20,000 854 4.60

Together, these data demonstrate the activated Ras selective growthinhibitory activity of the compounds of this invention. This is incontrast to the current clinically used inhibitors of proteins withinthe Ras signaling cascade that exhibit no selectivity or selectivity forcells lacking activated Ras.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and “at least one” andsimilar referents in the context of describing the invention (especiallyin the context of the following claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The use of the term “at least one”followed by a list of one or more items (for example, “at least one of Aand B”) is to be construed to mean one item selected from the listeditems (A or B) or any combination of two or more of the listed items (Aand B), unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1-32. (canceled)
 33. A method of selectively inhibiting a Ras-mediatedbiological process in vivo in a human, which process is growth of anaturally occurring tumor cell harboring constitutively activated Ras,which method comprising steps of: a) identifying a selectiveRas-inhibitory compound of formula II or selective Ras-inhibitory saltthereof, b) identifying a human within whom is growing a naturallyoccurring tumor cell harboring constitutively activated Ras, and c)administering in vivo to said human identified according to step b) aRas-inhibiting effective amount of said selective Ras-inhibitorycompound of formula II or selective Ras-inhibitory salt thereofidentified according to step a):

wherein: R and R₀ are hydrogens; n is 0, 1 or 2; Y and Y′ together isdouble-bonded oxygen; R₁, R₂, and R₃ are independently selected fromhydrogen, hydroxyl, halogen, alkyl, alkoxy, and alkylmercapto; R₄ and R₇are hydrogens; R₈ is selected from hydrogen, alkyl, and alkoxy; R₁₂ andR₁₆ are hydrogens; R₁₃ is alkoxy; R₁₄ is hydroxyl; R₁₅ is selected fromhydroxyl and alkoxy; X is NR′R″, where R′ is selected from the groupconsisting of phenyl, phenylalkyl and heterocycloalkyl where theheterocycle of the heterocycloalkyl is selected from pyridinyl,piperidinyl, piperazinyl, pyrrolidinyl and morpholinyl, wherein any ofthe cyclic structures of R′ may be unsubstituted or substituted with oneor more of halo, haloalkyl, alkoxy, hydroxyl, amino, alkylamino,dialkylamino and sulfonamido; and R″ is hydrogen; wherein all alkylsubstituents and all alkyl parts of all alkoxy, alkylmercapto,alkylamino, dialkylamino, phenylalkyl and heterocycloalkyl substituentscontain from one to six carbon atoms; and wherein said selectiveRas-inhibitory compound or selective Ras-inhibitory salt thereof in stepa) is a compound of formula II or a salt thereof that causes selectiveRas inhibition; and, wherein said identifying a selective Ras-inhibitorycompound or selective Ras-inhibitory salt thereof in step a) employs anin vitro assay for selective Ras inhibition performed on at least onecompound or salt of formula II or salt thereof; and wherein said invitro assay for selective Ras inhibition measures selective,preferential or specific inhibition of activated Ras, or inhibition ofat least one Ras-mediated process in a cell or tissue harboringconstitutively activated Ras relative to said process in a cell ortissue lacking activated Ras; and wherein said Ras-mediated processmeasured in said in vitro assay for selective Ras inhibition in saidcell or tissue is selected from growth, proliferation, invasiveness,metastasis, drug resistance and radiation resistance of a cell or tissueharboring activated Ras relative to said process in a cell or tissuelacking activated Ras; and wherein said identifying a human within whomis growing a naturally occurring tumor cell harboring constitutivelyactivated Ras in step b) employs an in vivo or in vitro assay of saidhuman's own tissue, cells, blood or tumor for an abnormal, mutant orhyperactive ras gene, Ras protein or Ras-mediated process selected fromgrowth, proliferation, invasiveness, metastasis, drug resistance andradiation resistance of a cell or tissue harboring constitutivelyactivated Ras relative to said process in a cell or tissue lackingactivated Ras; and, wherein said Ras-inhibiting effective amount in stepc) is an amount required to achieve in vivo in or on said tumor cellgrowing in said human a selective Ras-inhibiting level of said selectiveRas-inhibitory compound of formula II or selective Ras-inhibitory saltthereof to cause selective inhibition of growth of said tumor cellharboring constitutively activated Ras, relative to a cell within saidhuman that lacks activated Ras or an activating mutation of a ras gene.34. The method of claim 33, wherein said identifying a selectiveRas-inhibitory compound of formula II or selective Ras-inhibitory saltthereof in step a) is done using an in vitro assay performed on tissue,cells, blood, or tumor of said human identified in step b) of claim 1.35. The method of claim 33, wherein said identifying a selectiveRas-inhibitory compound of formula II or selective Ras-inhibitory saltthereof in step a) is done using an in vitro assay performed onestablished human tumor cell lines in cell culture to determine aselectivity index of a compound of formula II or salt thereof forinhibition of a cell harboring constitutively activated Ras relative toa cell lacking activated Ras.
 36. The method of claim 35, wherein saidselectivity index is determined and expressed as the numerical ratio ofthe concentration of said compound of formula II or salt thereof tocause in vitro 50% growth inhibition of a cell, selected from COLO 205,Caco-2, and HT-29, lacking activated Ras to the concentration of saidcompound to cause in vitro 50% growth inhibition of a cell, selectedfrom A549, HCT116, and SW480, harboring activated Ras.
 37. The method ofclaim 36, wherein said selectivity index is determined to be at leastten.
 38. The method of claim 37, wherein said selectivity index isdetermined to be at least one hundred.
 39. The method of claim 33,wherein R₁ and R₃ are hydrogen and R₂ is selected from hydroxyl,halogen, alkyl, alkoxy, and alkylmercapto.
 40. The method of claim 39,wherein R₂ is selected from halogen and alkoxy.
 41. The method of claim40, wherein R₂ is selected from fluorine and methoxy.
 42. The method ofclaim 33, wherein R₈ is selected from alkyl and alkoxy.
 43. The methodof claim 42, wherein R₈ is alkyl.
 44. The method of claim 33, whereinthe heterocycle of the heterocycloalkyl is selected from pyridinyl,piperidinyl, piperazinyl, and pyrrolidinyl.
 45. The method of claim 33,wherein R′ is phenyl or phenylalkyl, optionally substituted on thephenyl or the phenyl of the phenylalkyl by one or more halogen atoms.46. The method of claim 43, wherein said selective Ras-inhibitorycompound is selected from:(Z)—N-(2-(dimethylamino)ethyl)-2-(1-(4-hydroxy-3,5-dimethoxybenzylidene)-5-methoxy-2-methyl-1H-inden-3-yl)acetamide(003),(Z)-2-(1-(4-hydroxy-3,5-dimethoxybenzylidene)-5-methoxy-2-methyl-1H-inden-3-yl)-N-(pyridin-3-yl)acetamide(004),(Z)-2-(1-(4-hydroxy-3,5-dimethoxybenzylidene)-5-methoxy-2-methyl-1H-inden-3-yl)-N-(pyridin-3-ylmethyl)acetamide(009),(Z)-2-(5-fluoro-1-(4-hydroxy-3,5-dimethoxybenzylidene)-2-methyl-1H-inden-3-yl)-N-(pyridin-2-ylmethyl)acetamide(010),(Z)-2-(5-fluoro-1-(4-hydroxy-3,5-dimethoxybenzylidene)-2-methyl-1H-inden-3-yl)-N-(pyridin-3-ylmethyl)acetamide(011),(Z)—N-benzyl-2-(5-fluoro-1-(4-hydroxy-3,5-dimethoxybenzylidene)-2-methyl-1H-inden-3-yl)acetamide(015),(Z)-2-(1-(4-hydroxy-3,5-dimethoxybenzylidene)-5-methoxy-2-methyl-1H-inden-3-yl)-N-(pyridin-2-ylmethyl)acetamide(028),(Z)-2-(1-(4-hydroxy-3,5-dimethoxybenzylidene)-5-methoxy-2-methyl-1H-inden-3-yl)-N-phenylacetamide(034),(Z)-2-(5-fluoro-1-(4-hydroxy-3,5-dimethoxybenzylidene)-2-methyl-1H-inden-3-yl)-N-phenylacetamide(035), or the corresponding Z- or E-isomer thereof, or selectiveRas-inhibitory salt thereof.
 47. The method of claim 33, wherein saidconstitutively activated Ras is encoded by an activating mutation of aras gene.
 48. The method of claim 43, wherein said tumor cell is a lungtumor cell, a colon tumor cell, or a pancreas tumor cell.
 49. The methodof claim 33, wherein said tumor cell is a neurofibroma tumor cell. 50.The method of claim 33, wherein said tumor cell is drug resistant, orradiation resistant.
 51. A method wherein, in addition to the stepsa)-c) of the method of claim 33, at least one other agent, which is nota compound of formula II or salt thereof, selected from an anticanceragent and radiation, is administered in vivo in step c).